maryam amra jordaan - ETD-db - University of the Free State

THE SYNTHESIS OF AN INTERNAL
STANDARD FOR BICALUTAMIDE
MARYAM AMRA JORDAAN

THE SYNTHESIS OF AN INTERNAL
STANDARD FOR BICALUTAMIDE
Thesis submitted in fulfillment of the requirements for the degree
Master of Science in Chemistry
in the
Department of Chemistry
Faculty of Agricultural and Natural Science
at the
University of the Free State
Bloemfontein
by
MARYAM AMRA JORDAAN
Supervisor: Prof. J.H. van der Westhuizen
Co-Supervisor: Prof. B.C.B. Bezuidenhout
January 2008

ACKNOWLEDGEMENTS
I would like to thank the almighty ALLAH for the strength and perseverance that he has
given me to complete this study.
I wish to express my sincere gratitude to the following people:
My husband Yasar and daughter Aminah for their support, patience and love during
difficult circumstances;
Prof. J.H. van der Westhuizen as supervisor and mentor for the invaluable assistance and
guidance that he has given me;
Dr. S.L. Bonnet for her professional research guidance;
Prof. B.C.B. Bezuidenhout as co-supervisor for assistance, advice and encouragement;
The NRF, THRIP, UFS for financial support;
Mrs. Anette Allemann, Prof. T. van der Merwe and Dr. Gideon Steyl for the recording of
MS, IR data and 19 F NMR spectra;
Mrs. Alice Stander for the editing of this thesis;
To my mother and father as well as the rest of the Jordaan family for their encouragement
To the Amra, Ibrahim, Dada and Docrat families for their support;
To the staff and fellow postgraduate students in the Chemistry department for their
assistance especially, Anke.
M. Amra Jordaan

Summary/Opsomming
Abbreviations
1. Chapter 1: Literature Survey
Table of Contents
1.1 Introduction 2
1.2 Pharmacology of bicalutamide 3
1.3 Synthesis of bicalutamide 6
1.4 Synthesis of enantiopure (R)- and (S)-bicalutamide 10
1.5 Synthesis of derivatives of (R,S)-bicalutamide 15
1.6 Chemical and biochemical transformations of bicalutamide 23
1.7 References 24
2. Chapter 2: Results and Discussion
2.1 Introduction 25
2.2 Internal standards 26
2.3 Synthesis of Internal Standards 27
2.4 Synthesis of Internal Standards for bicalutamide 28
2.5
2.4.1 Synthesis of deuterated bicalutamide 28
2.4.2 De novo synthesis of structural analogues of bicalutamide 30
2.4.3 Modifications to bicalutamide 33
19
F NMR spectroscopy 48
2.6 Conclusions 50
2.7 Future work 51
2.8 Structure elucidation
2.8.1 2-Oxo-N-phenylbutanamide (84) 52
2.8.2 N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-oxobutanamide (86) 53
2.8.3 Starting material (bicalutamide) (1) 54
1

2.8.4 3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methyl-N-[4-methyl-3-
(trifluoromethyl)phenyl]propanamide (94)
2.8.5 4-[3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methylpropylamido]-2-
(trifluoromethyl)benzamide (96)
2.8.6 1-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(4-
fluorophenylsulfonyl)-2-methyl-1-oxopropan-2-yl 3-nitrobenzoate (98)
2.8.7 (Z)-N-[4-Cyano-3-(trifluoromethyl)phenyl] -3-(4-fluorophenylsulfonyl)-
2.8.8
2.9
2-methylacrylamide (99)
4-Amino-2-ethoxybenzonitrile (100)
References
3 Chapter 3: Experimental
3.1 Chromatographic Techniques 64
3.1.1 Thin-Layer Chromatography 64
3.1.2 Centrifugal Chromatography 64
3.1.3 Column Chromatography 65
3.1.4 Spraying Reagents 65
3.2 Gas Chromatography 65
3.3 Spectroscopic Methods 66
3.3.1 Nuclear Magnetic Resonance Spectroscopy 66
3.3.2 Mass Spectrometry 66
3.3.3 Infrared Spectrometry 67
3.4 Physical properties measurement 67
3.4.1 Melting Point 67
3.5 Photochemical Reactions 67
3.6 Chemical Methods 67
3.6.1 Methylation with diazomethane 67
56
58
59
60
62
63

3.7 Synthesis
3.7.1 a) Synthesis of 2-oxo-N-phenylbutanamide (84) 68
3.7.1 b) Attempted synthesis of 2-((4-fluorophenylsulfonyl)methyl)-2-
hydroxy-N-phenylbutanamide (85)
3.7.2 a) N-(4-cyano-3-(trifluoromethyl)phenyl)-2-oxobutanamide (86) 70
3.7.2. b) Attempted synthesis of N-[4-cyano-3-(trifluoromethyl)phenyl]-2- [(4-
fluorophenylsulfonyl)methyl]-2-hydroxybutanamide (87)
3.7.3 Extraction of Bicalutamide; N-[cyano-3-(trifluoromethyl)phenyl]-3- [(4-
fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide (1) from
Casodex ®
3.7.4 3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-N-[4-methyl-3-
(trifluoromethyl)phenyl]propanamide (94)
3.7.5 Synthesis of 4-(3-[4-Fluorophenylsulfonyl)-2-hydroxy-2)-2-
methylpropanamido]-2-(trifluoromethyl)benzamide (96)
3.7.6 Attempted synthesis of (Z)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-
fluorophenylsulfonyl)-2-methylacrylamide (99)
3.7.7 Synthesis of 1-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(4-
fluorophenylsulfonyl)-2-methyl-1-oxopropan-2-yl 3-nitrobenzoate (98)
and Synthesis of (Z)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-
fluorophenylsulfonyl)-2-methylacrylamide (99)
3.7.8 Synthesis of 4-amino-2-(ethoxy)benzonitrile (100) (I) 77
3.7.9 Synthesis of 4-amino-2-(ethoxy)benzonitrile (100) (II) 78
69
71
71
72
73
74
75

APPENDIX
NMR PLATES 1-10
IR PLATES 1-3
GC PLATES 1

SUMMARY
(R,S)-Bicalutamide [N-(4-cyano-3-trifluoromethylphenyl)-α-methyl-α-hydroxy-β-(4-
fluorophenylsulfonyl)propanamide], sold as Casodex ® , is the leading antiandrogen
currently used to treat prostate cancer. It binds to androgen receptors and blocks
cancer growth.
This work aims to develop internal standards for the bio-analytical component of
clinical trials that are required to detect bicalutamide and derivatives. An internal
standard is added to the body fluid sample (mostly blood) at the beginning of the
sample work up at about the same concentration of the analyte to be quantified. An
ideal internal standard has a similar extraction recovery and a similar retention time in
HPLC. For quantification with mass spectrometry it should have a difference of at
least 3 mass units from the analyte and a similar ionization response. The internal
standard is used to calibrate the total ion current of the metabolite.
We did not have access to deuterium labeled starting materials and investigated
structural analogues as an alternative strategy to obtain internal standards. De novo
synthesis of structural analogues failed because we could not deprotonate the methyl
sulfone in the presence of aromatic amides. We ascribed this to incomplete disclosure
in the patented methods.
Treatment of bicalutamide with palladium on activated charcoal under the right
conditions did not give the usually produced amide but gave smooth reduction of the
C≡N group to a CH3 group. This unusual reduction gave ready access to a good
internal standard in good yield.
Elimination of the tertiary aliphatic hydroxy group of bicalutamide would give an
alkene with similar polarity that could serve as an internal standard. Acid catalysis

using 1M HCl or p-toluene sulfonic acid failed, but treatment of bicalutamide with
H2SO4 in benzene gave hydrolysis of the nitrile to an amide. This provides a second
internal standard in good yield.
Bicalutamide did not react with weak base. Strong base such as LDA led to fission of
the aliphatic moiety and isolation of aromatic sulfone and amide fragments.
Derivitization of the tertiary aliphatic hydroxy group of bicalutamide with 3-
nitrobenzoyl chloride gave a benzoyl ester that allows facile thermal elimination of
nitrobenzoic acid at 40 ºC to form an alkene. This represents a third potential internal
standard. The NOESY experiment proves that the alkene has a Z-configuration. This
indicates that the pro-R aliphatic hydrogen of bicalutamide was eliminated
stereoselectively via a syn-periplanar cyclic transition state.
Efforts to eliminate the hydroxy group of bicalutamide photolytically at 300 nm in
ethanol yielded an unexpected replacement of the aromatic CF3 group with an ethoxy
group. We could not find a similar transformation in the literature and believe it to be
a novel reaction.
We used 19 F NMR to prove the absence or presence of fluorine and CF3 moieties in
the products. We also used the magnitude of 13 C- 19 F coupling constants in proton
decoupled 13 C spectra for accurate resonance assignment and structure elucidation.
We will test these novel analogues of bicalutamide in cancer bioassays and use them
as internal standards for the quantification of bicalutamide and analogues in body
fluids.

OPSOMMING
(R,S)-Bicalutamide [N-(4-siano-3-trifluorometielfeniel)-α-metiel-α-hydroksi-β-(4-
fluorofenielsulfoniel)propaanamied], wat verkoop word onder die handelsnaam
Casodex ® , word beskou as een van die belangrikste anti-androgene middels wat tans
gebruik word om prostraatkanker te behandel. Dit bind met androgene reseptore en
inhibeer kanker groei.
Hierdie navorsing beoog om interne standaarde te ontwikkel vir die bio-analitiese
komponent van kliniese studies wat vereis word om bikalutamied en derivate te
registreer. ‘n Interne standard word aan die begin van die opwerk van die
liggaamsvloeistofmonsters (meestal bloed) bygevoeg teen ongeveer dieselfde
konsentrasie as die analiete wat gekwantifiseer moet word. Die ideale interne
standaard ekstraëer teen ‘n soortgelyke konsentrasie en het ongeveer dieselfde
retensietyd in HPLC as die analiet. Vir kwantifisering met massaspektrometrie moet
daar ‘n verskil van ten minste 3 massa-eenhede en ‘n soortgelyke ionisasie reaksie in
vergelyking met die analiet wees. Die interne standaard word gebruik om die totale
ioon-vloei van die metaboliete te kalibreer.
Ons het nie toegang gehad tot gedeutereerde uitgangstowwe nie en het strukturele
analoë ondersoek as ‘n alternatiewe strategie om interne standaarde daar te stel. De
novo sintese van strukturele analoë het gefaal aangesien ons nie die metielsulfone kon
deprotoneer in die teenwoordigheid van aromatiese amiede nie. Ons het dit toegeskryf
aan onvolledige inligting in die gepatenteerde metodes.
Behandeling van bikalutamied met palladium op geaktiveerde koolstof onder die
regte toestande het nie die gewone amied geproduseer nie, maar het geredelik die
C≡N-groep na ‘n CH3-groep gereduseer. Hierdie ongewone reduksie gee in ‘n goeie
opbrengs gerieflike en maklike toegang tot ‘n interne standaard.

Eliminasie van die tersiêre alifatiese hidroksielgroep van bikalutamied behoort alkene
met ‘n polariteit soortgelyk aan dié van die analiet te lewer wat as interne standaard
kan dien. Suurkatalise (HCl (1 M) of p-tolueensulfoonsuur) het gefaal, maar
behandeling van bikalutamied met H2SO4 in benseen het die nitriel na ‘n amied
gehidroliseer, wat ‘n tweede interne standaard in ‘n goeie opbrengs lewer.
Bikalutamied het nie met ‘n swak basis reageer nie, maar sterk basisse soos LDA het
tot splyting van die alifatiese moeïeteit en isolasie van aromatiese sulfone en
amiedfragmente gelei.
Derivatisering van die tersiêre alifatiese hidroksielgroep van bikalutamied met 3-
nitrobensoïelchloried het ‘n bensoïelester gelewer wat teen 40 o C fasiele termiese
eliminasie van nitrobensoësuur toelaat om ‘n alkeen te vorm. Dit verteenwoordig ‘n
derde potensiële interne standaard. NOESY eksperimente het bewys dat die alkeen ‘n
Z-konfigurasie het, wat toon dat die pro-R alifatiese waterstof (en nie die pro-S
alifatiese waterstof) van bikalutamied stereochemies geëlimineer is via ‘n syn-
periplanêre sikliese transisie toestand.
Pogings om die hidroksielgroep van bikalutamied fotolities teen 300 nm in etanol te
elimineer, het ‘n onverwagse vervanging van die aromatiese CF3-groep met ‘n
etoksiegroep teweeggebring. Ons kon geen soortgelyke transformasie in die literatuur
opspoor nie en glo dat dit ‘n unieke en nuwe reaksie is.
Ons het van 19 F KMR gebruik gemaak om die teenwoordigheid of afwesigheid van
fluoriede en CF3- moeïeteite in ons produkte aan te dui. Die groottes van die 13 C- 19 F
koppelingskonstantes in ons proton-ontkoppelde 13 C KMR spektra lei tot die akkurate
toekenning van resonansies en struktuuropklaring.

Ons sal hierdie nuwe analoë van bikalutamied in kanker bio-assesering toets en as
interne standaarde gebruik vir die kwantifisering van bikalutamied en analoë in
liggaamsvloeistowwe.

Abbreviations
The following abbreviations were used to describe the solvent systems and
reagents used in this study:
A acetone
BuLi butyllithium
CDCl3 chloroform-d
DCM dichloromethane
EtOAc ethyl acetate
H hexane
K2CO3 potassium carbonate
LAH lithium aluminum hydride
NH3 ammonia
TEA triethylamine
THF tetrahydrofuran
NaOH sodium hydroxide
H2SO4 sulphuric acid

1.1 Introduction
Chapter 1
Literature Review
Carcinoma of the prostate is the third leading cause of death for US men, after
heart disease and stroke. It is estimated that one in six men will develop prostate
cancer in the United States. The American Cancer Society estimated that in 2005
alone, 232 090 new cases were diagnosed with prostate cancer and from these 30
1, 2
350 deaths occured.
(R,S)-Bicalutamide [N-(4-cyano-3-trifluoromethylphenyl)-α-hydroxy-α-methyl-β-
(4-fluorophenylsulfonyl)propanamide] (1) sold as Casodex ® is the leading
antiandrogen currently used to treat prostate cancer. 3 Casodex ® was first launched
in 1995 as a combination treatment (with surgical or medical castration) and
subsequently launched as monotherapy for the treatment of earlier stages of the
disease. 4
The growth of prostate cancer is stimulated by androgens, the male sex hormones.
The pioneering work of Huggins and Hodges in 1941 showed the hormone
dependence of this tumor. Androgen deprivation thus becomes the main treatment.
This is achieved by castration, either alone or in combination with an antiandrogen
5, 6
such as bicalutamide.
The median survival of patients with metastatic androgen-independent prostate
cancer is one year without the use of second-line hormonal therapy such as
bicalutamide. Although most men with androgen-independent prostate cancer
eventually develop symptoms related to metastases, often the first indication of
disease progression is an asymptomatic increase in serum prostate-specific antigen
(PSA) levels noted during routine surveillance. 7
2

F
1.2 Pharmacology of bicalutamide
O
S
O
OH CH 3
1
3
O
H
N CF 3
Most deaths from prostate cancer are caused by metastases. Although localized
cancer can be treated with a good prognosis, metastatic prostate cancer resists
conventional anti-cancer drugs and develops into incurable androgen refractory
prostate cancer. This has been attributed to the genetic, biological, biochemical
and immunologic diversity of prostate cancer cells. Research now concentrates on
the mechanism of tumerogenesis and metastases in the hope of overcoming the
2, 8
heterogeneity of the disease.
Testosterone is the major endogenous ligand that complexes with the androgen
receptor. It is synthesized in the testes (85%) and the adrenal cortex (15%). During
the early stages of prostate cancer androgen activation of the androgen receptor
exacerbates the disease and stimulates hyperplasia of the prostate. Removal of the
testes is the simplest and most frequently used method to remove the stimulatory
effect of testosterone on the growth of prostate cancer metastases. The adrenal
2, 8
gland however remains and only partial androgen deprivation is achieved.
CN

Antiandrogens that bind to the androgen receptor and thereby block androgen
action, from whatever source, have the potential to affect maximum androgen
withdrawal. Early treatment of the disease during the so-called hormone receptive
stage with a non-steroidal androgen receptor prostate-selective antagonist now
2, 8
benefits patients with a 99.8% 5-year survival rate.
Cyproterone acetate 2 was the first clinically used antiandrogen (early 1960s). It is
effective in most patients and produces fewer side effects than estrogens. The
steroidal nature of the drug is probably responsible for most of its remaining side
effects including cardiovascular effects, adverse effects on serum lipoproteins,
effects on carbohydrate metabolism (and diabetes) and severely depressed libido.
O
H 2C CH 3
Cl
2
4
AcO
CH3 Flutamide 3 was the first non-steroidal antiandrogen. It is a pure antiandrogen that
does not exhibit any of the side effects associated with steroidal drugs. It rarely
causes loss of libido. It represents an important improvement but still causes side
effects in some patients including gastrointestinal intolerance and gynecomastia.
Irreversible liver function abnormalities can be serious. Flutamide antagonises the
action of androgen at the hypothalamus and pituitary gland. This leads to an
increase in luteinizing hormone (LH) that stimulates androgen secretion by the
testes and progressively higher doses of flutamide is required if the testes had not
been removed. The active metabolite of flutamide, hydroxyflutamide (4) has a
biological half-life of 5.2 hours. This requires the drug to be administered three
times daily.
H 3C
O

O 2N
O 2N
F 3C
CF 3
3
4
Nilutamide 5 is structurally related to flutamide and replaced it. It has an improved
biological half-life (two days) that allows once daily administration. It causes side
effects, including alcohol intolerance, interstitial pneumosis and problems with
light-dark adaptation. It was eventually replaced by bicalutamide as the treatment
of choice.
O 2N
F 3C
5
5
H
N
N
H
O
N
O
O
O
H
CH 3
NH
CH
OH
CH 3
CH 3
CH 3

Bicalutamide is a peripherally selective non-steroidal anti-androgen that was
selected from 2000 compounds specifically designed for anti-androgen activity. It
has a long half-life (once daily oral administration), does not stimulate LT
production (does not require removal of the testes and can consequently be used as
a monotherapy), does not interfere with libido and is generally better tolerated
than flutamide or nilutamide. Its peripheral selectivity (little effect on serum LH
and testosterone) is due to poor penetration of the blood-brain barrier. 8
Bicalutamide has also been indicated as treatment for familial male-limited
precocious puberty. It leads to decreased facial acne and pubic hair. It achieved a
marked decrease in growth velocity and skeletal advancement in treated patients. 9
The (S)-isomer of bicalutamide is metabolized much faster than the (R)-isomer
and is thus eliminated faster. The (S)-isomer consequently has a shorter half-life,
put more stress on the liver and requires larger doses to be effective. It would be
advantageous, particularly for patients with hepatic impairment, to administer the
(R)-isomer only. Astrazeneca has recently patented the formulation of (R)-
3, 10
bicalutamide.
Analytical methods to quantify levels of bicalutamide in body fluids has been
described in detail by Rao and co-workers. 10
1.3 Synthesis of (R,S)-bicalutamide
Tucker and co-workers 6 reported the original synthesis of racemic bicalutamide
(1) in 1988. It is comprised of four steps and has an overall yield of 50% (Scheme
1).
6

NC
F 3C
F
F
6
F
O
10
Scheme 1
NH 2
O
O
S
O
S
O
H
N
H
N
SH
OH
Conditions: (a) CH3CONMe2, 2,6-Dimethylphenol; (b) CH3CCl3, mCPBA ; (c) NaH, THF; (d)
CH2Cl2, mCPBA .
7
+
8
9
11
CH 3
O
OH CH 3
1
a)
b)
c)
d)
O
CF 3
CF 3
O Cl
CN
CN
7
H
N CF 3
CN
H
N CF 3
CN

In 2002 James and Ekwuribe 11 described an improved synthesis of the
enantiomeric mixture in two steps with an overall yield of 73%, (Scheme 2).
Thionyl chloride was found to catalyse the nucleophilic addition of the aniline (6)
derivative (a poor nucleophile due to two electron withdrawing groups on the
aromatic ring) to pyruvic acid to give the α-keto acid 13 in a good yield (80%).
Sodium hydride was used to deprotonate the methyl sulfone to form the dimer.
Use of a stronger base (pentylmagnesium bromide or n-butyllithium) improved the
desired deprotonation.
CN
H 2N CF 3
F
F
O
Scheme 2
O
O O
S
O O
Conditions: (a) DMA, SOCl2; (b) BuLi, THF .
6
14
S
1
+
H
N
13
8
a)
b)
OH
O
H
N
O
12
O
CF 3
CN
OH
CF 3
CN

Chen and co-workers 12 synthesized (R,S)-bicalutamide from the less expensive 4fluoro-2-trifluoromethylbenzonitrile
15. They described the first use of the
methylacrylamide anion in nuleophilic aromatic substitution. The three-step
synthesis was reported to give an overall yield of more than 90% (Scheme 3).
F
F
15
O
CH 2
CF 3
CN
O
O
O O
S
Scheme 3
H
N
H
N
1
+
8
9
Conditions: (a)(i) H2O/HCl, (ii) 2.8 eq. NaH/DMF, 97%; (b) H2O2/(CF3CO)2O, CH2Cl2, 98%; (c) 4-
FC6H4SH/NaH/THF; (d) H2O2/(CF3CO)2O, CH2Cl2, 97%.
9
CH 3
a)
b)
c)
d)
OH
O
CH 3
H
N
O
16
CF 3
CN
CF 3
CN
NH 2
CF 3
CN

1.4 Production of enantiopure (R) - and (S)-bicalutamide
Torok and co-workers 13 developed a new route for the production of racemic
(R,S)-bicalutamide (Scheme 4). High-performance liquid chromatographic
methods were developed for the enantioseparation of (R,S)-bicalutamide.
F 3C
NC
F 3C
NC
F 3C
NC
10
F
H
N
F
O
SO 2Na
17
H
N
O
Scheme 4
H 3C OH
O
a)
b)
c)
F 3C
+
NC
10
CH 2Cl
O
H3C OH
H
N S
O
1
+
O
S
O
O
H 3C OH
O
(19, side product)
OH
H
N
H 3C OH
O
(18, side product)
F
CH 2OH
Conditions: (a) NaOH in acetone, 68%; (b) AcOH in MeOH, 43%; (c) tetrabutylammonium bromide,
MeOH, 62% (18) and (19) isolation by chromatography.

Fujino and co-workers 14 published a synthesis of enantiopure (R)-bicalutamide.
They started with an epoxide mixture of (R)-20 and (S)-20. Treatment with an
engineered Bacillus subtilis transforms only the (R)-isomer and left the (S)-isomer
unconsumed. Subsequent treatment of the resulting mixture of (R)-21 and
unconsumed (S)-20 with dilute H2SO4 transforms unconsumed R-enantiomer to
(R)-21 with 100% ee conversion (Scheme 5a). They also developed a method to
synthesize (R)-bicalutamide from (R)-21 (Scheme 5b) and (R)-20 (Scheme 5c).
BnO
Scheme 5a
O
BnO OH
OH
11
BnO
BnO
BnO OH
OH
+
(R)-20 a)
(S)-20
+
50%
b)
50%
(R)-21
(S)-20, unconsumed
(R)-21, 100% ee
Reagents and conditions: (a) B. subtilis epoxide hydrolase, 30 ºC, 7 days, conv 50%; (b) dilute
H2SO4, room temperature.
O
O

F
O
BnO N
H
HO 23
OH
HO
O O
S
O
Scheme 5b
BnO OH
BnO
14
HO
(R)-21
HO
22
N
H
24
(R)-1
a)
b)
c)
d)
e)
CO 2H
12
H 2N CN
Reagents and conditions: (a) TEMPO, NaClO, NaClO2, MeCN-buffer, 35 0 C, 24 h, 97%; (b) SOCl2,
THF, DMAP, room temperature, 5days; (c) Ac2O, pyridine, 83% ; (d) DDQ, hv (352 nm, 15 W),
MeCN, 85%; (e) K2CO3, MeOH, 85%;
CF 3
CF 3
CN
6
CN
CF 3

Scheme 5c
O
F S
OBn
F
O
(R)-20
O
13
O
S
O
HO
OBn
F S OBn
25
26
27
a)
b)
c)
d)
HO
HO
F S
N
H
O
O
O HO CH 3
(R)-1
e)
Reagents and conditions: (a) 4-fluorothiophenol , NaH, THF, room temperature, 90 min, 93%; (b)
H2O2, AcOH, 60 ºC, 24h; (c) H2, Pd-C, EtOH, room temperature, 48h, 91% from 25; (d) TEMPO,
NaClO, NaClO2, MeCN-buffer, 93%; (e) SOCl2, THF, 4-cyano-3-trifluoromethyl-aniline, room
temperature, 5 days, 91%.
CO 2H
CF 3
CN

In 2002, James and Ekwuribe 3 published an improved version of (R)-bicalutamide
synthesis based on naturally occurring (S)-citramalic acid (30) as starting material
(Scheme 6). This route has the advantage of one less step and the use of natural
(S)-citramalic acid in more cost effective synthesis.
F
HO
O
H 3C OH
OH
Scheme 6
HO
28
O
29
b)
N+
O -
F
F
30
SH
Br
S OH
32
H 3C
11
OH
O
S
O
S
O
a)
H 3C
14
O
H
(R)-33
e)
O
OH
O
H 3C OH
O
O
CBr 3
H
N
F
O
H 3C
O
H
O
O
CBr 3
SH
CN
H2N CF3 6
Reagents and conditions: (a) Tribromo acetalaldehyde, H2SO4; (b) DCC, CBrCl3; (c) NaOH, i-PrOH ;
(d) SOCl2, DMA; (e) mCPBA.
31
(R)-1
H
N
CF 3
CF 3
c)
d)
CN
CN

1.5 Synthesis of derivatives of (R,S)-bicalutamide
Tucker and co-workers 6 successfully prepared analogs of bicalutamide Table 1
using the synthetic route illustrated in Scheme 1. These compounds were also
tested for anti-androgen activity.
F 3C
H 3C
H
N
Table 1: Analogues of Bicalutamide
Compound R 2
34
35
36
37
38
39
40
41
42
1
43
44
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CN
CN
CN
CN
CN
R 3
3-Cl
2-Cl
4-F
4-F
4-NO2
4-CN
4-CH3O
4-CH3S
4-F
4-F
4-Cl
4-CH3S
R 2
O
1
15
OH
X
R 3
X Formula
S
S
S
SO2
S
S
S
S
S
SO2
S
S
C17H14ClF3N2O4S
C17H14ClF3N2O4S
C17H14F4N2O4S
C17H14F3N2O6S
C17H14F3N3O6S
C18H14F3N3O4S
C18H17F3N2O5S
C18H17F3N2O4S2
C18H14F4N2O2S
C18H14F4N2O4S
C17H14ClF3N2O2S
C19H17F3N2O2S2
Patil and co-workers 15 synthesized a derivative of bicalutamide (Scheme 7) in
which the sulfone group was replaced by oxygen 48. The hydroxy group of
compound 48 was protected using tert-butyldimethylsilyl trifluoromethane
sulfonate with 2,6-lutidine in dichloromethane. N-methyl derivatives of compound
49 were prepared using a CsF-Celite/alkyl halide/acetonitrile combination. During

the N-methylation a 1, 4-N→O migration of the disubstituted phenyl ring was
observed to yield compound 53.
H 2N
Br
H 3C
c)
CF 3
Scheme 7
+
HO
CN
H3C OH
6 45
OH
O
H
N
HO F
d)
e)
47
46
NC
NC
NC
F 3C
F 3C
F 3C
CF 3
CN
O
CH 3
N
16
b)
NH
NH
O
H 3C
O
H 3C
49
50
O
H 3C
Br
O CH 3
48
O
Si
O
Si
O
OH
a)
H
N
O
O F
9
CF 3
CN
O F
F

F 3C
H 3C H 3C OH
N O
O 51
NC F
F 3C
NC
F 3C
NC
H
H3C OCH3 N O
O
H3C OH
H
N O
H 3C N
H
O
+
53
+
O
C
+
52
+
48
Reagents and conditions: (a) SOCl2, TEA, THF; (b) anhyd. K2CO3, anhyd. acetone; (c) anhyd.
K2CO3; (d) TBDMSOTf, 2,6-lutidine, CH2Cl2; (e) CH3ICsF-Celite, ∆.
In 2005 Nair and co-workers 16 synthesized iodo analogs of bicalutamide (Scheme
8) that contained a radioactive isotopic label ( 125 I) to allow the radioimaging of
prostate cancer. These compounds were obtained by replacing the trifluoromethyl
and sulfonyl groups with iodine and oxygen respectively, as well as the
introduction of chirality at the stereogenic centre. In 2006 Nair and co-workers 17
17
O
CN
O
CF 3
F
F
F

also synthesized chiral oxazolidinedione derived bicalutamide analogs (Scheme
9a and b).
Scheme 8
H 2N
I NO 2
NC
I
H
N COOH
HO Br
H
OH
56 57
NC
I N
H
58
NC
I N
H
59
N
H
O
18
HO
OH
NC
54 55
a)
b)
60
c)
d)
e)
f)
I NH 2
O
O
O
O
51
OH
O
Br
NH-tBoc
NH-tBoc

NC
Me 3Sn
NC
*I
NC
N
H
N
H
*I N
H
NC
*I N
H
O
O
61
62
O
O
g)
h)
63
i)
64
19
OH
OH
OH
OH
O
O
O
O
NH-tBoc
NH-tBoc
Reagents and conditions: (a) (1) NaNO2, H2SO4, CuCN, NaCN, (2) HCl, SnCl2⋅2H2O, EtOH; (b) (1)
methacryloyl chloride, NaOH, acetone, (2) NBS, DMF, (3) HBr; (c) SOCl2, THF; (d) K2CO3, acetone;
(e) K2CO3, 2-propanol; (f) Pd(PPh3)4, hexamethyl ditin, toluene; (g) NaI (Na 125 I), chloramines T,
MeOH; (h) acetyl chloride, absolute EtOH; (i) chloroform, thiophosgene, NaHCO3; *I = I or 125 I.
NH 2
NCS

O
O
N
H
65
N
N
H 3C
H
COOH
a)
CH 3
H
COOH
O
H
Br
Scheme 9a
20
O
OH
66 68
N
O
H 3C
b) f)
67
O
67
O
O
H
H 3C
69
O
Br
H
O
e)
Br
O
OH
CBr 3
Br

O
N
CH 3
O
c) g)
H
S
O
70 71
F d)
21
OH OH
O H 3C
Reagents: (a) Methacryloyl chloride, NaOH, acetone; (b) NBS, DMF; (c) NaOH, 4’fluorobenzenethiol,
2-propanol; (d) concd HCl; (e) 24% HBr; (f) tribromoacetalaldehyde, concd
H2SO4; (g) (1) NaOH, 4-fluorobenzenethiol, 2-propanol, (2) conc. HCl.
S
F

O 2N
O 2N
Scheme 9b
F3C NH2F3C NCS
72 73
HO OH
O 2N
F 3C N
F 3C
O 2N
O
O H 3C
F 3C
CH 3
S
74
h)
S
N O O
S
..
Ag HO
75
N
76
22
O
O
F
O 2N
CH 3
S
O
CH3 S
O
F
O
77
Reagents: (h) NaHCO3, thiophosgene, CH2Cl2; (i) acetonitrile, silver trifluoroacetate, triethylamine.
i)
F
F

1.6 Chemical and biochemical transformations of bicalutamide
No references for the in vivo chemical degradation of bicalutamide to its
metabolites or in vitro chemical transformations could be found.
23

1.7 References
1. Ng, R. A.; Guan, J.; Alford, V. C.; Lanter, J. C.; Allen, G. F.; Sbriscia, T.;
Linten, O.; Lundeen, S. G.; Sui, Z. Bioorg. Med. Chem. Lett., 2007, 17, 784-
788.
2. Rashid, H. H.; Nelson, J.; Koenemane, K. S. Urol Oncol-Semin Ori., 2006, 24,
243-245.
3. James, K. D.; Ekwuribe, N. N. Tetrahedron., 2002, 58, 5905-5908.
4. www.astrazeneca.com/productbrowse/5_82.aspx (16:27 on 13/09/2007).
5. Fitzpatrick, J. M.; Newling, D.; Vela-Navaretta, R. Eur. Urol. Suppl. 1. 2002,
39-43.
6. Tucker, H.; Cook, J. W.; Chesterson, G. J. J. Med. Chem., 1988, 31, 954-959.
7. OH, W. K. Urol. Suppl. 3A., 2002, 60, 87-93.
8. Furr, B. J.; Tucker, H. Urol. (Suppl. 1A)., 1996, 47, 13-25.
9. Kreher, N. C.; Pescovitz, O. H.; Delameter, P.; Tiulpakov, A.; Hochberg, Z. J.
Ped., 2006, 149, 416-420.
10. Rao, R. N.; Raju, A. N.; Nagaraju D. J. Pharmacol. Biomed. Anal., 2006, 1-7.
11. James, K. D.; Ekwuribe, N. N. Synthesis., 2002, 850-852.
12. Chen, B. C.; Zhao, R.; Cove, S.; Wang, B.; Sundeen, J. E.; Salvati, M. E.;
Barrish, J. C. J. Org. Chem., 2003, 68, 10181-10182.
13. Torok, R.; Bor, A.; Orosz, G.; Lukảs, F.; Armstrong, D. W.; Pẻter, A. J.
Chrom. A., 2005, 1098, 75-81.
14. Fujino, A.; Asano, M.; Yamaguchi, H.; Shirasaka, N.; Sakoda, A.; Ikunaka,
M.; Obata, R.; Nishiyama, S.; Sugai, T. Tetrahedron. Lett., 2007, 48, 979-983.
15. Patil, R.; Li, W.; Ross, C. R.; Kraka, E.; Cremer, D.; Mohler, M. L.; Dalton, J.
T.; Miller, D. D. Tetrahedron Lett., 2006, 47, 3941-3944.
16. Nair, V. A.; Mustafa, S. M.; Mohler, M. L.; Yang, J.; Kirkovsky, L. I.; Dalton,
J. T.; Miller, D. D. Tetrahedron Lett., 2005, 46, 4821-4823.
17. Nair, V. A.; Mustafa, S. M.; Mohler, M. L.; Dalton, J. T.; Miller, D. D.
Tetrahedron Lett., 2006, 47, 3953-3955.
24

2.1 Introduction
Chapter 2
Results and Discussion
The registration of a new medicine requires clinical trials. These are not complete
without quantitative bio-analysis of body fluid samples. Regulatory authorities
(e.g. the Food and Drug Administration or FDA in the United States) require
extensive studies on the concentration of the new medicine (analyte) in human
blood, it’s half life (how long it stays in the human body) and products that it is
metabolized to (metabolites) by enzymes in the blood, before they will allow it to
be registered. To determine the concentration of medicine in human body fluids a
unique internal standard is required for each new medicine.
PAREXEL is a leading global bio/pharmaceutical services organization that helps
clients expedite time-to-market through their development and launch services.
These include a broad range of clinical development capabilities, integrated
advanced technologies, regulatory affairs consulting, and commercialization
services.
Over the past 25 years, PAREXEL has developed significant expertise to assist
clients in the worldwide pharmaceutical, biotechnology and medical device
industries with the development and launch of their products in order to bring safe
and effective treatments to the global marketplace for the patients who need them.
For drug and device developers, clinical research is a complex, uncertain, yet
highly important and necessary endeavor. While innovative ideas are put to the
test, substantial investments weigh in the balance and regulators oversee
everything. These researchers need a proven partner that can assemble and deploy
all the necessary capabilities required to fulfill the various activities of clinical
research.
25

FARMOVS-PAREXEL has a bio-analytical service laboratory in Bloemfontein.
This company analyses blood and other biological fluids for pharmaceuticals and
pharmaceutical metabolites to assist local and international pharmaceutical
companies to register new medicines in South Africa and overseas.
Reference materials of internal standards and other metabolites are required to
validate their analytical methods. These are not always commercially available.
The capacity to manufacture these internal standards and metabolites enables
FARMOVS-PAREXEL not only to expand its services into trials from which it
has been excluded but also to offer services not available internationally. The
ability to custom synthesize internal standards provides FARMOVS-PAREXEL
with a unique competitive advantage in this already sophisticated and competitive
field.
This project aims to develop internal standards for the bio-analytical component of
clinical trials that are required to register bicalutamide, an anticancer drug (see the
literature review for more information on the pharmacology and chemistry of
bicalutamide) and analogues.
2.2 Internal standards
An internal standard is used for calibration and validation in quantitative bioanalytical
chemistry. The internal standard is added to the body fluid sample
(mostly blood) at the beginning of the sample work up at about the same
concentration of the analyte to be quantified. It controls variability during
extraction, sample preparation and quantification during mass spectrometry
analysis.
FARMOVS-PAREXEL uses mass spectrometry (total ion current of a specific
mass fragment of the analyte under investigation). This represents a sophisticated
and selective method of quantification. By choosing the right fragment to monitor,
26

interference from other metabolites and impurities in a complex body fluid sample
can for all practical purposes be eliminated. The ideal internal standard should
behave exactly like the metabolite under investigation during extraction from body
fluids and sample preparation but should be distinguishable during the
quantification process.
An ideal internal standard for quantification with mass spectrometry is an
isotopically labeled form of the molecule with a difference of at least 3 mass units
from the analyte that is to be quantified. An isotopically labeled internal standard
will have a similar extraction recovery, ionization response in ESI mass
spectrometry and a similar retention time in HPLC but differs in its recorded mass.
Polarity and pKa plays an important role in these parameters.
If isotopically labeled internal standards are not available, structural analogues
may be used. Of importance is that it has the same minimum of three mass unit
difference that is required for isotopically labeled versions and that it co-elutes
with the compound to be quantified. A chlorinated version of the parent molecule
often has the same chromatographic retention time and differs sufficiently in mass.
Hydroxylated (+16 amu) and demethylated (-14 amu) versions should be avoided
as the human body often manufactures these analogues in unknown quantities
from the parent compound as part of its normal metabolic processes. The human
body normally metabolizes pharmaceuticals to more polar metabolites to allow
1, 2
secretion into urine.
2.3 Synthesis of internal standards (mass spectrometry quantification)
Three possible strategies were investigated to synthesize internal standards:
1) De novo synthesis of an isotopic isomer. The isomer is identical in all
aspects except the mass difference due to the replacement of, for instance,
27

hydrogen with deuterium or 16 O with 18 O. To achieve the required mass
unit difference, three hydrogens or two oxygens must be replaced.
2) De novo synthesis of a closely related analogue. Replacement of, for
instance a methyl group with an ethyl or chlorine with a fluorine atom may
yield the desired internal standard. The assumption is that there will be only
a very small change in polarity and behaviour of the molecule during the
extraction process.
3) Modification of the analyte itself to a closely related analogue with a
difference of at least three mass units.
2.4 Synthesis of internal standards for bicalutamide
2.4.1 Synthesis of deuterated bicalutamide
The synthesis of deuterated bicalutamide depends on the commercial
availability of deuterium labeled starting materials. According to the review of
synthetic procedures in Chapter 1, the following starting materials should be
suitable.
28

F 3C
NC
D
D
NH 2
D
D
F
Figure 1
D
D
29
O O
S
D
D 3C
78 79 80
Two factors affected our decision not to proceed with this strategy:
1) The deuterated starting materials (78, 79 and 80) are not readily
available commercially. Previous efforts in our laboratory to exchange
H with D using D2O obtained from published methods gave poor
results.
2) A regulatory requirement is that isotopically labeled internal standards
should contain less than 1% unlabeled product to ensure that only the
analyte is recorded. Marginal isotopic purity of commercially available
deuterated starting materials (99%) are often not high enough to
achieve this. We have developed a strategy to overcome this problem
by starting with two separately labeled building blocks. This, however,
increases the cost and complexity of any potential synthetic method.
O
O
OD

2.4.2 De novo synthesis of structural analogues of bicalutamide
Most of the published syntheses of bicalutamide uses the following starting
materials (literature review, chapter 1):
1) 4-Amino-2-(trifluoromethyl)benzonitrile (6)
2) Phenylmethyl sulfone (14)
3) Pyruvic acid (12)
For the De novo synthesis of structural analogues many possibilities exist as any
analogue of 6, 14 or 12 could be used.
Two options were investigated:
1) We decided to use 2-ketobutyric acid 81 and aniline 83 instead of pyruvic
acid 12 and 4-amino-2-(trifluoromethyl)benzonitrile 6. The use of aniline
in a model reaction saved costs on the use of expensive material. The
synthesis of the amide 84 was successful but the coupling between 14 and
84 failed to produce compound 85. However the starting materials were
recovered (Scheme 13)
2) The other option was to replace pyruvic acid 12 with 2-ketobutyric acid 81
in order to transform the 2-methyl group to a 2-ethyl group. This would not
change the polarity significantly and would add 14 g/mol in the mass
spectrometry. The rest of the starting materials remained the same. The
synthesis of the amide 86 was successful. The coupling between the
sulphone 14 and 86 did not succeed and compound 87 was not isolated
(Scheme 14).
30

The coupling required deprotonation of the sulphone 14 and we suspect that the
deprotonation of the amide interfered.
Gas chromatography was used to monitor the formation of 82 as this compound is
volatile and TLC was unsuitable (Chapter 3). This enabled us to optimize our
conditions in improving the yields of 85 and 87. (GC Plate 13).
Scheme 13
F
DCM
83
NH 2
O O
S
14
O
81
O
82
H
N
84
O
SOCl 2
O
THF
31
O
BuLi
OH
Cl
O
H
OH
N S
O
Triethylamine
O
O
85, NOT ISOLATED
F

F 3C
F
NC
F 3C
NC
F 3C
NC
6
DCM
NH 2
O O
S
14
Scheme 14
O
81
O
82
H
N
86
32
O
SOCl 2
O
THF
O
BuLi
OH
Cl
O
H
OH
N S
O
Triethylamine
O
O
87, NOT ISOLATED
F

F
O
S
O
CH 3
Scheme 15
BuLi
F
14 14a
In view of the fact that our other efforts to modify bicalutamide directly (described
below) had succeeded and we had already produced three internal standard
analogues, we decided not to pursue our de novo syntheses further. Our decision
was also influenced by the realization that the published syntheses of bicalutamide
were based on patents. It is a well known fact that inventors often do not give full
disclosure of the finer details of their invention in order to frustrate attempts to
improve on their invention. From previous experience we suspect that essential
finer details were omitted.
2.4.3. Modifications of bicalutamide
Inspection of bicalutamide revealed the following functional groups that could be
modified:
1) A fluorine atom which could be replaced by hydrogen or another
halogen.
2) A C≡N group, which could be reduced or oxidized.
3) An SO 2 group, which could be reduced.
4) An OH group, which could be derivatized or eliminated.
5) An amide group that could be alkylated.
33
O
S
O
CH 2

We decided to explore the following avenues:
A. Reduction of the C≡N group
B. Elimination of the OH group.
C. Methylation of the OH group.
A. Reduction of the C≡N group.
Reduction of a nitrile group to an amine is a well-known reaction. 3 Selectivity
and efficiency in the reduction of nitriles are important for the preparation of
amino derivatives in organic synthesis. This reduction is important in industry.
Numerous reagents and methods have been developed to reduce aromatic
nitrile groups, such as:
1) Metal/acid reduction
2) Catalytic hydrogenation
3) Electrolytic reduction
4) Homogeneous catalytic transfer hydrogenation
5) Heterogeneous catalytic transfer hydrogenation
34

All these methods, however, have limitations, such as:
1) The metal/acid system lacks selectivity and requires a strong acidic
medium.
2) Catalytic hydrogenation employs highly diffusible, low molecular weight,
flammable hydrogen gas.
3) In electrolytic reduction yields are low and there is a lack of practical utility
in academic institutions.
4) Homogenous catalytic transfer hydrogenation requires expensive reagents
as catalysts. Work up and isolation of the products are not trivial.
5) Heterogeneous catalytic transfer hydrogenation employs metals like
palladium, platinum and ruthenium. These catalysts require stringent
precautions, because of their flammable nature in the presence of air and
hydrogen. Because of the small quantities of the internal standard required
for bio-analysis, cost of the catalyst is not important for this application.
Raney nickel is routinely used as a catalyst in the field of catalytic hydrogenation
as well as in the field of heterogeneous catalytic transfer hydrogenation. 4
Reduction of the cyano group to an alkylamine is the general transformation.
However, further reduction to a methyl group has only rarely been reported.
Brown and co-workers 5 reported this rare reduction using ammonium formate as
the hydrogen source in the presence of a 10 % palladium on carbon catalyst
(Scheme 16). This hydrogenation occurred at room temperature over a 24 hour
period. The reaction rate is influenced by the amount of catalyst employed and
temperature. Equal weights of catalyst to nitrile have to be used to ensure a
smooth conversion to the corresponding methyl derivative.
35

CN
OH
88
Scheme 16
10% Pd-C / HCOONH 2, r.t.
Bumgardner and co-workers 6 reported the reduction of primary aliphatic amines to
alkanes (90) with HNF2 (91). It was postulated that R−N=N−H is an intermediate,
so that the reaction proceeds through the carbocation (92) (Scheme 17).
Scheme 17
36
CH 3
OH
3RNH2 + HNF3 2R NH3F + N2 + R-H
90 91 92 93
An indirect means of achieving the same result is conversion of the primary amine
to sulfonamide RNHSO2R' and treatment of this with hydroxylamine-O-sulfonic
acid. The same intermediate, R−N=N−H is postulated in this case.
Brieger and Nestrick 7 investigated C≡N reduction to CH3 and reported various
reaction conditions such as nature of the donors, solvent effects, and the effects of
temperature on reduction.
We decided to investigate heterogeneous catalytic transfer hydrogenation because
pressure equipment and the supported metals such as palladium and nickel were
readily available to us as only a small quantity of the internal standard were
required.
89

Sulfones are resistant to reducing agents and were not expected to interfere in the
reduction of the nitrile group. It seems that α-anion formation makes the sulfone
inert with reducing agents such as LiAlH4. 8
Our first step was to reduce the cyano group with Raney nickel using an autoclave
at high temperature and pressure. This method however was unsuccessful
probably due to the poor quality of the Raney nickel (W-11) used.
We then decided to use palladium on activated charcoal and the result was
unexpected as the C≡N group was smoothly reduced to a CH3 group to afford
compound 94 (Scheme 18). This reaction is reminiscent to the palladiumcatalyzed
hydrogenolysis of benzyl alcohols, yielding toluene. 9 Amine 95 was not
isolated but served as an intermediate to the formation of 94.
F 3C
H 3C
F 3C
NC
O
OH
H
N S
O
94
O
Scheme 18
H
N
F
O
1
Pd-C
EtOH
24 hrs
37
OH
F 3C
H 2NH 2C
O
S
O
F
O
OH
H
N S
O
95, NOT ISOLATED
A full structure elucidation on 94 is given in section 2.8, but the following salient
features of the unexpected compound are important:
O
F

1) The mass spectrum is in full agreement with this assignment; (M + ) -
(C18H17NO4F4S ) in negative electron ion mode: m/z = 419.0895 compared
to M + (C18H18N2O4F4S): m/z = 435 for the amine 95.
2) In the 1 H NMR spectrum a new doublet was observed at δ 2.45 (J = 1.4
Hz), which integrated for three protons. This peak was assigned to the
benzylic CH3 because the COSY shows a coupling between CH3 and H-5".
Benzylic four bond coupling splits the CH3 resonance into a doublet (J =
1.4 Hz). A CH2 group attached to an amine 95 would integrate for two
protons only.
3) In the 13 C NMR spectrum the C≡N group at δ 114.98 was replaced by a
new resonance at δ 18.81 which was assigned to the CH3 group. (CH2NH2
would be expected at about δ 46.0). 8
4) The HMBC experiment shows the three-bond coupling (correlation)
between C-3" and CH3 as well as a three-bond coupling between C-5" and
CH3.
These features confirm the postulated structure. This compound is an ideal internal
standard because there is a difference in mass of about 11 mass units and the
polarity difference is acceptable.
B. The elimination of the OH group.
Bicalutamide has a tertiary hydroxy group, which is normally difficult to
eliminate. The following options to eliminate the OH group were investigated to
overcome this problem:
a) Acid catalysis
b) Base catalysis
38

c) Thermal elimination
d) Photochemistry
a) Acid Catalysis
Acid catalysis was attempted (employing 1M HCl in ethanol). The reaction was
refluxed for a few days, however, no reaction took place. Refluxing bicalutamide
in benzene (bp 110 ºC) with p-toluenesulfonic acid also produced no results. The
addition of H2SO4 to the benzene reaction mixture resulted in isolation of 96
(Scheme 19), i.e. hydrolysis of the nitrile group to an amide.
F 3C
NC
H 2N
F 3C
O
Scheme 19
O
OH
H
N S
O
H 2SO 4
39
O
O
OH
H
N S
O
1
96
p-Toluene sulfonic acid
Benzene
O
F
F

A full structure elucidation on compound 96 is given in section 2.8 but the
following salient features of the amide are important:
1) The amide is characterized by an (M-H) - mass of 447.0640 in electron ion
negative mode.
2) The amide is further characterized by an additional carbonyl group at δ
168.03 in the 13 C NMR spectrum.
3) The IR spectrum shows the characteristic amide stretching frequency which
consists of two stretching frequencies at 3448 cm -1 (plate 6h).
The product 96 was inert to methylation with diazomethane. This proves that
hydrolyses to a carboxylic acid did not take place. The acid would require a mass
of (M + ) - 448 in the negative mode and (M + ) 449 [M + H] + , these peaks, however,
were not observed in the mass spectrum.
b) Base Catalysis
To eliminate the OH group we tried a strong hindered base, lithium
diisopropylamide (LDA). This resulted in cleavage of bicalutamide and the
isolation of 6 and 14 (Scheme 20). This is probably due to the presence of trace
amounts of water. Water reacts with LDA to give lithuim hydroxide which
hydrolyses the amide bond of bicalutamide. A retro-aldol reaction results in the
isolation of 14.
40

F 3C
NC
F 3C
NC
6
H
N
LDA
THF
Scheme 20
H3C OH
O O
O
NH 2
+
1
F
100 0 C
2hrs
41
14
S
O O
A weaker base such as K2CO3 was used but it could not deprotonate the OH
group, and no reaction took place.
c) Thermal elimination
Heating of bicalutamide up to 200 ºC under argon did not produce any
transformations.
To transform the OH group into a better leaving group we derivatized it with 3nitrobenzoylchloride
97. Stirring bicalutamide at room temperature with
nitrobenzoylchloride 10 in DCM with catalytic amounts of pyridine gave the 3nitro-benzoyl
ester 98.
Heating of the ester 98 to 40 ºC yielded the target alkene 99 via elimination of 3nitrobenzoic
acid (Scheme 21). The same result was achieved if 98 were not
isolated and the reaction mixture was heated directly.
S
F

Cl
F 4C
NC
O
F 3C
NC
97
F 3C
NC
Scheme 21
H
N
NO 2
O
42
OH
O
S
O
O
H
N S
O
1
O
98
Pyridine
DCM
DMAP
O
O
Heat (40 0 C)
H 3C H
NH
(Z)-99
The thermal elimination of the nitrobenzoyl group is expected to take place via a
syn-periplanar cyclic transition state 98a.
F
S
O
O
O
NO 2
F
F

F 3C
O
CN
NH
The two protons on the CH2 group adjacent to the sulfonyl group are
diastereotopic. Elimination of the pro-R hydrogen will give a Z alkene and of the
pro-S hydrogen will give an E alkene. 11 The presence of a NOESY between the
CH3, H (plate 7d) proves that the pro-R hydrogen was eliminated to give an alkene
with a Z-configuration (99a).
F 3C
NC
O
O
43
98a
H S
F
O
H
H 3C H
NH
NOESY
A probable explanation for the observed stereoselectivity and the absence of any
E-alkene is that a hydrogen bond between the SO2 group and the N-H of the amide
stereoselectively favours the transition state that the selective removal of the pro-R
hydrogen to form the Z-configurated alkene (99b).
99a
F
S
O
NO 2
O
O
O

C. Photochemistry
F 3C
NC
O
O
O
N H
99b
NO 2
Examples of elimination of OH using photochemistry have been reported. 12
Irradiation of bicalutamide at 300 nm, however, unexpectedly yielded compound
100. This product requires hydrolysis of the amide bond to yield the aniline
derivative 6, followed by replacement of the CF3 group by an ethoxy group in a
typical photocatalyzed SN reaction. The trifluoromethyl group is normally
chemically inert. It is also resistant to many metabolic transformations. 13 The high
carbon-fluorine bond energy makes fluorine a poor leaving group in nucleophilic
substitution reactions. This indicates that the CF3 group was directly substituted
(Scheme 22). The nature of the substituent (OCH2CH3) indicates an ionic
mechanism via a π,π*-excited singlet. Radical substitution would have yielded a
CH(OH)CH3 derivative. 12 During photochemical reactions excitation of π, π*singlets
are normally associated with ionic reactions and π,π*-triplets with radical
mechanisms
We repeated the photolytic reaction starting with 4-amino-3-
(trifluoromethyl)benzonitrile (Scheme 23) and obtained the same product 100
under the same photolytic conditions in 50 % yield. We could not find any
H
S
44
O
O
F

eference to this transformation in the literature and believe it to be a new reaction.
We will investigate the synthetic potential of this further.
F 3C
NC
H
N
EtOH
Scheme 22
H3C OH
O O
O
F 3C NH 2
NC
1
6
hv (300nm)
45
S
hv (300nm)
H 3CH 2CO NH 2
NC
100
F

F 3C
NC
H 3CH 2CO
NC
Scheme 23
EtOH
6
46
100
NH 2
hv (300nm)
A full structure elucidation is given in section 2.8. The following features however
are the most important:
1) The absence of carbon-fluorine couplings in the 13 C NMR spectrum and
fluorine resonances in the 19 F NMR spectrum indicate the absence of
fluorine present.
2) The NOESY experiment demonstrates a coupling between H-2 and the
ethyl CH2 as well as coupling between H-2 and the ethyl CH3. (Plate 8c).
3) The NOESY experiment also shows the coupling of the NH2 group to H-3
and H-5 and proves that the NH2 group is flanked by these protons. (Plate
8e)
4) Mass spectrometry agrees with the replacement of CF3 (M + (C8H5N2F3):
m/z = 186.1) with OCH2CH3 (M + (C9H10N2O): m/z = 163.0874).
NH 2

D. Methylation
Our first attempt to methylate the aliphatic hydroxy group of bicalutamide
involved the use of K2CO3 as base and the addition of MeI in DMA. This reaction
gave no results as the base used could not deprotonate the OH in bicalutamide. We
then used sodium as a base and then afterwards MeI was added. Purification by
chromatography was difficult but from cursory NMR it was observed that
cleavage of bicalutamide occurred. This is attributed to a typical retro-aldol
reaction.
Methylation with LDA and MeI gave an intractable mixture. We assumed that
methylation of the amide gives rise to rotational isomers which gives complicated
NMR spectra. The amide N-CO bond has a partial double-bond character arising
from the contribution of resonance structure 102 (Scheme 24) to the ground state
of the amide. A large barrier to rotation around the amide N-CO bond is provided.
This leads to geometrical and magnetical nonequivalence of the N-substituents. 14
O
R N
R 2
R 1
Scheme 24
47
R N
101 102
O
R 2
R 1

2.5 19 F NMR Spectroscopy
The 19 F nucleus (I = ½, natural abundance 100%) has 83% of the sensitivity to
NMR spectroscopy detection compared to that of a 1 H nucleus. As such it is a
useful tool to detect the presence of fluorine in a molecule. Most modern probeheads
can be tuned to the 19 F frequency because of the proximity of the resonance
frequencies of 1 H and 19 F. No special equipment other than a 19 F preamplifier is
needed for a standard experiment.
Introduction of fluorine into a potential drug can produce a wide range of
biological effects, ranging from complete metabolic inertness to high specificity of
binding to a particular protein receptor site. Because fluorine rarely occurs in
natural compounds, it has become a very useful tool to investigate and monitor
biological processes (eg. enzyme product identification and monitoring a complex
biological mixture). Because of fluorine’s high sensitivity, it has also become a
tool to determine the concentration of fluorine in solutions (quantitative NMR).
The concentration of fluorinated molecules in a range of 10 µM can be
conveniently determined in about 15 minutes. 15
Due to the large range in fluorine chemical shifts (from -131 to + 129 relative to
CF3COOH in ppm) no single fluorine-containing compound can be used
experimentally as a universal reference compound (c.f. tetramethylsilane in 1 H and
13
C NMR spectroscopy). Any fluorine containing compound that resonates in the
region of the internal standard can be used as reference. We used trifluoroacetic
acid which resonates at about δ -78.7 relative to TMS. An NMR tube insert was
used to keep the reference and the fluorine containing compound under
investigation apart and to avoid the effect of dilution on the chemical shift of the
reference compound (This enabled us to use 99% trifluoroacetic acid as a
reference). Fluorine chemical shifts are significantly more sensitive than proton
chemical shifts to concentration and temperature of the sample and these should be
specified.
48

C-F couplings are visible in proton decoupled (CPD) 13 C NMR spectra because
only 1 H is decoupled and not 19 F. The size of the couplings between fluorine and
carbon is very useful in determining the position of fluorine in a molecule and is
given in (Scheme 25). It gives an indication of the size of the coupling (JCF) as a
function of the number of bonds (x) between carbon and fluorine. 16
CF 3
Figure 2
δ (CF) 124.5: 1 JCF = 272
δ (C1) 131.0: 2 JCF =32
δ (C2) 125.3: 3 JCF = 4
δ (C3) 128.8: 4 JCF = 1
δ (C4) 131.8: 5 JCF = 0
49
F
δ (C1) 163.3: 1 JCF = 245.1
δ (C2) 115.5: 2 JCF = 21.0
δ (C3) 130.1: 3 JCF = 7.8
δ (C4) 125.0: 4 JCF = 3.2
We observed the aromatic CF3 group in our products and starting materials at
about -63 ppm and the aromatic F substituent at about -106 ppm (in acetone-d6)
(Table 2).
Table 2. Chemical shift of 19 F in bicalutamide, derivatives of bicalutamide
and fragments of bicalutamide
Compound CF3 (δ) 4'-F (δ)
1 -62.60 -106.31
6 -63.00
14 -106.7
86 -62.7
94 -61.74 -106.61
96 -59.57 -106.68
98 -62.82 -105.63
99 -62.74 -105.28

2.6 Our investigation achieved the following:
1. Four new derivatives of bicalutamide have been synthesized.
2. Three of these derivatives are suitable internal standards for
quantitative bio-analysis of body fluid samples.
3. These derivatives are novel and will be submitted for testing in anticancer
bioassays.
4. a) A simple method for C≡N reduction to CH3 in bicalutamide has
been developed.
b) A method for hydrolysis of C≡N to CONH2 in bicalutamide has
been established.
c) A reaction for the facile elimination of OH to form an alkene
under mild conditions has been developed.
5. A new photochemical method for the substitution of CF3 by an
ethoxy group in aromatic amines has been developed.
6. We used the 13 C- 19 F coupling as a useful tool to identify and
elucidate the structures of all the derivatives of bicalutamide.
7. We used fluorine NMR to validate the presence of a CF3 and F
group in a reaction product.
50

2.7 Future Work
We plan the following:
1) Methylation of bicalutamide with diazomethane indicates that a very slow
reaction takes place. Heating is not permissible as diazomethane will
evaporate and an autoclave cannot be used because of the explosion danger
associated with diazomethane. We will reinvestigate this reaction.
2) We will investigate the synthetic potential of the photolytic replacement of
the aromatic CF3 with OCH2CH3 starting with model compounds. This
reaction appears novel as we could not find a literature reference.
3) The synthesis of bicalutamide and bicalutamide derivatives from the
deprotonation of 4-fluorophenyl methyl sulfone and amide addition will be
attempted again with different bases and under anhydrous conditions.
4) We will test our novel derivatives in anti-cancer screening and as antiandrogens.
51

2.8 Structure Elucidation
Comprehensive interpretations of the 1 H, 13 C, APT, COSY, HSQC, HMBC, IR
and NOESY spectra of bicalutamide and its reaction products are given below:
2.8.1 2-Oxo-N-phenylbutanamide (84)
A. Mass spectrometry confirmed assignment of structure 84, found (ES) [M+H] + ,
178.0870 (C10H11NO2 + H + ) required m/z 178.0868.
B. The salient features in the 1 H NMR spectrum are (Plate 1a):
1) According to the integrals in the 1 H NMR spectrum (H-2'/ H-6') and (H-
3'/ H-5') in the aniline ring resonates at δ 7.65 (d, J = 8.0 Hz), δ 7.37 (t,
J = 8.0 Hz), and integrated for two protons each.
2) H-4' at δ 7.18 (t, J = 8.0 Hz) integrated for one proton.
3) The C-2 (CH 2) resonance is observed as a quartet at δ 3.05 (J = 7.3 Hz)
and integrated for two protons.
4) The C-3 (CH3) resonated as a triplet at δ 1.16 and integrated for
three protons.
C. The salient features in the 13 C NMR spectrum are (Plate 1b):
1) (C-2'/C-6') and (C-3'/ C-5') resonates at δ 119.8 and δ 129.2, respectively.
These resonances were confirmed by correlation with (H-2'/ H-6') and (H-
3'/ H-5') at δ 7.65 and δ 7.37 in the HSQC (Plate 1d).
2) C-4' resonates at δ 125.2. This was confirmed by correlation with H-4' at δ
7.18 in the HSQC (Plate 1d).
52

D. The salient features in the COSY experiment (Plate 1c):
Cross peaks in the COSY confirmed the expected aliphatic coupling between CH2 δ 3.05 (J = 7.3 Hz) and CH3 at δ 1.16 (J = 7.3 Hz).
E. The HMBC and APT data did not add any additional information that could
not be obtained from the other methods.
2.8.2 N-[4-(cyano-3-(trifluoromethyl)phenyl]-2-oxobutanamide (86)
A. Mass spectrometry confirmed assignment of structure (86), found (ES) [M-H + ] -
269.0541 (C 12H 9F 3N 2O 2 - H + ) required m/z 269.0538.
B. The salient features in the 1 H NMR spectrum are (Plate 2a):
1) According to the integrals in 1 H NMR spectrum, H-2, H-6, H-5 in the AMX
system resonated at δ 8.55 (J = 1.9 Hz), δ 8.37 (J = 1.9 Hz,) and δ 8.09 (J = 8.5
Hz) respectively and integrated for one proton each.
2) The C-2 (CH 2) two doublets at δ 3.02 (J = 7.2 Hz) was clearly visible and
integrated for two protons.
3) The C-3(CH 3) resonated as a triplet at δ 1.10 and integrated for three protons.
C. The salient features in the 13 C NMR spectrum are (Plate 2b):
1) Assignment of C-2, C-5, C-6 at δ 117.82, δ 123.04 and δ 136.25
respectively was confirmed by HSQC (plate 2d).
D. The salient features in the COSY spectrum are (Plate 2e):
53

Cross peaks in the COSY confirmed the expected aliphatic coupling between CH2 at δ 3.02 (J = 7.2 Hz) and CH3 at δ 1.10 (J = 7.2 Hz).
E. The HMBC and APT did not add any additional information that
could not be obtained from the other methods.
2.8.3 Starting Material (bicalutamide) (1)
A. Mass spectrometry confirmed assignment of structure (1), found (ES) MW
(M + ) 430.38 (C18H14F4N2O4+H + ) requires m/z 430.
B. The salient features in the 1 H NMR spectra are (plate 3a):
1) The aliphatic CH 3 hydrogen resonance was observed as a singlet at δ 1.42.
2) The aliphatic CH 2 group resonance was observed as two geminal doublets
(J = 14.8 Hz) at δ 3.57 and δ 4.00.
3) H–2", H–6" and H–5" of the tri-substituted aniline moiety were easily
recognized as an AMX system at δ 8.28 (J = 2.0 Hz), δ 8.08 (J = 2.0 Hz, J
= 8.5 Hz), δ 8.7.89 (J = 8.5 Hz), respectively. No fluorine couplings were
observed.
4) The observed hydrogen resonances on the p-fluorophenyl sulphone ring
showed clear hydrogen fluorine couplings. The AA'BB' system at δ 7.86
and δ 7.16 is split by ( 3 J FH = 9.0 Hz, ortho coupling), (H-3', H–5', closest to
fluorine) and 4 JFH = 5.0 Hz (H-2', H–6', next to the sulfone group). Because
3
JFC and 3 JFC are both 9.0 Hz, H-3'and H–6' appeared as a triplet.
54

C. The salient features in the 13 C NMR spectrum (Plate 3b) are:
a) The aliphatic region
1) The amide carbonyl resonance was observed at δ 172.60.
2) The CH 3 resonance was observed at δ 26.52 (negative APT, plate 3d and it
correlated with CH 3 at δ 1.42 in HSQC, plate 3e).
3) The CH 2 at C–3 was deshielded by the sulfone group and the resonance was
observed at δ 62.74. This was confirmed by correlation with CH 2 at δ 4.1 and δ
3.7 in the HSQC (plate 3e).
4) The quaternary C–2 had a small intensity. It was deshielded by the adjacent
hydroxy carbonyl groups and the resonance was observed at δ 73.25 (positive
APT, plate 3d).
b) The aniline ring
1) The CF 3 carbon resonance was observed as a quartet at δ 122.25 (negative APT,
plate 3d) with the characteristic large 1 J FC = 273.0 Hz coupling. 1
2) The quartenary 3"- carbon, next to the CF 3 group resonance was observed at δ
132.02 with ( 2 J FC = 32.0Hz).
3) The C≡N group resonates at δ 114.98 which was characteristic of this functional
group. 15
4) C–2", C–5" and C–6" correlated with the proton AMX system in the HSQC (plate
3e) and the resonance was observed at δ 117.01 ( 3 J FC = 5.1), δ 135.51 and δ
122.29, respectively (negative APT, plate 3d).
55

5) C–1" and C–4" resonances were observed at δ 142.70 and δ 102.87 (J = 2.2 Hz),
respectively.
c) Phenyl sulfone ring
1) The C–4' resonance at δ 165.03 was characterized by 1 J FC = 253.0 Hz.
2) C–3' and C–5' resonance at δ 115.47 were symmetrical and correlated with H–3'
and H–5' at δ 7.16 in the HSQC spectra (plate 3e). They were also identified by
2 JFC = 22.9 Hz.
3) C–2' and C–6' resonated at δ 131.10 which correlated with H–2' and H–6' at δ
7.86 in the HSQC spectra (plate 3e). They were also identified by 4 J FC = 9.8 Hz.
4) The resonance observed at δ 136.91 ( 1 J FC = 2.0 Hz) was assigned to C–1'. The
proximity to the sulfone group explained the large deshielding. It appeared as a
positive resonance in the APT (plate 3d) which confirmed the quartenary nature.
2.8.4. 3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methyl-N-[4-methyl-3-
(trifluoromethyl)phenyl]propanamide (94)
A. Mass spectrometry confirmed assignment of structure 94, found (ES) [M-H] +
(C18H17NO4F4S+H + ) required m/z 419.0893, in negative electron ion mode.
B. The salient features in the 1 H NMR spectrum (Plate 4a):
The NMR spectrum was similar to that of bicalutamide, the starting material, with
the following exceptions:
1) The AMX system of the aniline ring has lost deshielding of about 0.8ppm. This
illustrated the reduction of the C≡N group to CH3. (Plate 4a).
56

2) The new doublet at δ 2.45 (J = 1.4 Hz) that appeared in the spectrum was assigned
to the aromatic CH3.
C. The salient features in the 13 C NMR spectrum (Plate 4b):
1) The new peak at δ 18.78 was assigned to the CH3 group.
2) The C≡N group at δ 114.98 ppm had disappeared.
3) C-4" attached to the C≡N group in bicalutamide had moved to δ 135.20 and the
resonance was observed as a doublet with ( 3 JCF = 3.1 Hz).
D. The salient features in the COSY spectrum are (Plates 4d and 4e):
1) The COSY data demonstrated a strong cross coupling between H-5" at δ 7.24 and
CH3 of C-4" at δ 2.45 (J = 1.4 Hz), benzylic coupling.
2) The CH3 of C-4" at δ 2.45 also showed weaker cross couplings with H-6" at δ 7.46
and H-2" at δ 7.68 on the aniline ring.
E. The salient features in the HSQC spectrum (plate 4f) are:
The HSQC data confirmed the direct correlation between the CH3 carbon δ 135.20 and
the CH3 hydrogen at δ 2.45.
F. The salient features in the HMBC spectrum(Plate 4g) are:
HMBC data illustrated the three bond coupling between C-3" at δ 129.37 and CH3 at δ
2.45 as well as a three bond coupling between C-5" and CH3. This confirmed the
attachment of CH3 to C-4" of the aniline moiety in place of the CN group.
H. The salient features in the IR (Plate 4h and Plate 5h) are:
57

The disappearance of the characteristic C≡N group at 2229.3 was illustrated in the IR
comparison with bicalutamide (1). 15
2.8.5 4-[3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methylpropanamido]-2-
(trifluoromethyl)benzamide (96)
A. Mass spectrometry confirmed assignment of structure (96), found (ES) [M-H] +
447.0640 (C18H17NO4F4S-H + ) required m/z 447.0638, in the negative electron
ion mode.
B. The salient features in the 1 H NMR spectrum (Plate 5a) are:
The 1 H NMR spectrum was similar to that of bicalutamide, the starting material.
C. The salient features in the 13 C NMR spectrum (Plate 5b) are:
The amide was further characterized by an additional carbonyl group at δ 168.03 in
the 13 C spectrum.
D. The salient features in the IR spectrum (Plate 6h) include:
The IR data showed the characteristic amide stretching frequency which consisted of
two stretching frequencies at about 3448 cm -1 . 15
2.8.6 1-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(4-
fluorophenylsulfonyl)-2-methyl-1-oxopropan-2-yl 3-nitrobenzoate (98)
A. The mass spectrum confirmed structure 98, found (ES) [M-H] - , 579.0650
(C25H17F4N3O2S - H + ) required m/z 579.0645.
58

B. The salient features in the 1 H NMR spectrum (Plate 6a) are:
In addition to the observed proton resonances from the two aromatic moieties, the
AMX system and the AB system, four additional protons were observed in the
aromatic region. These protons were assigned as H-2''' at δ 8.58 (J = 0.4 Hz, 1.6 Hz,
2.3 Hz), H-4''' at δ 8.42 (J = 1.1 Hz, 2.3 Hz, 8.2 Hz), H-6''' at δ 8.25 (J = 1.1 Hz, 1.6
Hz, 7.4 Hz) and H-5''' at δ 7.74 (J = 0.4 Hz, 7.5 Hz, 8.2 Hz), J = 0.4 Hz corresponds
with p-coupling.
C. The salient features in the 13 C NMR spectrum (Plate 6b) are:
1) There were two C=O resonances at δ 168.87 and δ 165.90. The benzoyl ester
C=O group was assigned to δ 165.90.
2) C-4' directly attached to fluorine also resonated in the same area as the C=O
at δ 165.90 as a doublet with coupling ( 1 J = 254.2 Hz).
3) The C-1" resonance was observed as a doublet at δ 142.27 ( 1 JCF = 3.4 Hz).
4) The CF3 resonance was observed as a quartet at δ 122.10 ( 1 JCF = 273.0 Hz).
5) C-NO2 resonated at δ 129.75 which was characteristic of this functional
group. 15
6) The aliphatic carbon resonances were observed at δ 22.40 (CH3), δ 57.07
(CH2) and δ 80.24 (C-2). These resonances were similar to bicalutamide.
7) The C-1''' resonance was observed at δ 147.77 and C-1" resonated at δ
142.26.
8) C-2" resonated at δ 117.40 ( 1 JCF = 4.6 Hz).
59

9) The C-3'/ C-5' resonance was observed as a doublet at δ 116.54 ( 2 JCF = 22.8
Hz). This assignment was confirmed by the coupling between H-3'/ H-5' at δ
7.3 and C-3'/ C-5' at δ 116.54 in the HSQC (plate 6e).
10) C-2'/ C-6' resonated as a doublet at δ 131.30 ( 3 JCF = 9.8 Hz). This assignment
was confirmed by the coupling between H-2'/ H-6' at δ 8.01 and.C-2'/ C-6' at
δ 131.30 in the HSQC (plate 6e).
11) The remaining six hydrogen attached aromatic carbons resonated at:
δ 117.46 (C-6")
δ 124.43 (C-2")
δ 127.53 (C-4''')
δ 129.81 (C-5''')
δ 135.34 (C-6''')
δ 135.61 (C-5").
These assignments were confirmed with HSQC (plate 6e).
D. The salient features in the COSY spectrum (Plate 6d)
1) The COSY data demonstrated the correlation between the four nitrobenzoyl
protons at δ 8.58 (H-2'''), δ 8.42 (H-4'''), δ 8.25 (H-6''') δ 7.74 (H-5''').
2.8.7 (Z)-N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenylsulfonyl)-
2-methylacrylamide (99)
A. The mass spectrum confirmed structure 99, found (ES) [M+H] + , 413.0587
(C18H12F4N2O3S+H + ) required m/z 413.0583.
B. The salient features in the 1 H NMR spectrum (Plate 7a):
The NMR spectrum was similar to that of bicalutamide, the starting material, with
the following exceptions:
60

1) The expected resonances of the protons in the AMX H–2", H–6" and H–5" of the
tri-substituted aniline ring was easily recognized at δ 8.3 (J = 1.9Hz), δ 8.2 (J =
1.9Hz, J = 8.6Hz), δ 8.0 (J = 8.6Hz), respectively.
2) The expected resonances of the protons in the AB system was observed i.e. H-2'
and H-6' resonated at δ 8.1 (J = 5.1Hz, 8.9Hz) and H-3' and H-5' resonated at δ 7.5
(J = 8.9Hz).
3) The CH3 group resonated as a doublet at δ 2.41 (J = 1.5Hz) due to the crosscoupling
with the vinylic proton.
4) The vinylic proton resonance was observed at δ 7.2 (J = 1.4Hz, 2.9Hz).
C. The salient features in the NOESY spectrum (Plate 7d):
1) The NOESY data demonstrated the expected resonances correlation between the
vinylic proton and the methyl group.
2.8.8 4-Amino-2-ethoxybenzonitrile (100)
A. The mass spectrum confirmed structure 100, found (ES) [M+H] + , 163.0874
(C9H10N2O + H + ) required m/z 163.0872.
B. The salient features in 1 H NMR spectrum (Plate 8a):
The characteristic features of the NMR spectra are the following:
1) H-5, H-2 and H-6 in AMX system resonated at δ 7.52 (J = 8.5 Hz), δ 7.36 (J = 2.5
Hz) and δ 6.94 (J = 2.5 Hz, 8.5 Hz), respectively.
2) The characteristic broad peak of NH2 resonated at δ 5.86.
61

3) The CH2 group resonates as a quartet at δ 4.37 (J = 7.1Hz) and CH3 resonates as a
triplet at δ 1.38 (J = 7.1Hz)
C. The salient features in the 13 C NMR spectrum (Plate 8b):
It is clear from this spectrum that there was no flourine present in the molecule.
D. The salient features in the NOESY spectrum (Plate 8c, d, e):
1) The NOESY demonstrated a weak coupling between H-2 at δ 7.36 and CH2 at δ
4.37 (Plate 8c) as well as coupling between H-3 and CH3 at 1.38 (Plate 8d).
2) The coupling of the NH2 group to H-3 at δ 7.36 and H-5 at δ 6.94 proved that -
NH2 lies between these two protons (Plate 8e).
62

2.9 References:
1. Houghton, P.J.; Raman, A. (1998) A. Laboratory Handbook for the
Fractionation of Natural Extracts, 1 st ed; Chapman & Hall, London, pp.
134-135.
2. Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. (1997) Practical HPLC Method
Development, 2 nd ed, John Wiley & Sons Inc., Canada, pp. 657-659.
3. Caddick, S.; Judd, D. B.; Lewis, A. K.; Reich, M. T.; Williams, M. R. V.
Tetrahedron., 2003, 59, 5417-5423.
4. Gowda, S.; Gowda, D. C. Tetrahedron. 2002, 58, 2211-2213.
5. Brown, G. R.; Foubister, A. Synth. Commun., 1982, 1036.
6. Bumgardner, C. L.; Martin, K. J.; Freeman, J. P. J. Am. Chem. Soc., 1963,
85, 97-98.
7. Brieger, G.; Nestrick, T. Chem. Rev., 1974, 74, 567-580.
8. Barton, D.; Ollis, W. D. (1979) Comprehensive Organic Chemistry (The
Synthesis and Reactions of Organic compounds), 1 st ed, Pergamon Press,
Oxford, 3, pp. 181-182.
9. Matsura, V.A.; V. V. Potekhin, V. V.; Ukraintsev, V. B. (1996) Russ J Gen
Chem., 2002, 72, No. 1, 105-109
10. Mao, L.; Sun, C.; Zhang, H.; Li, Y.; Wu, D. Anal. Chim Acta., 2004, 522,
241-246.
11. Clayden, J.; Greeves, N.; Warren, S.; Wothers, P. (2001) Organic
Chemistry. Oxford University Press Inc, United States, pp. 836 & 488.
12. Qian, X.; Lui, S. J. Fluoro Chem., 1996, 79, 9-12.
13. Han, Z. “Photochemistry of Pentoxifylline A Xanthine Derivative”, U.F.S,
M.Sc Thesis, 2007.
14. Kim, Y. J.; Park, Y.; Park, K. K. J. Mol. Struct., 2006, 783, 61-65.
15. Berger, S.; Braun, S. (2004) 200 and more NMR Experiments, 3 rd edn,
Wiley-vch, Germany, pp. 336.
16. Affolter, C. (2000) Structure Determination of Organic Compounds,
Springer, pp. 113, 126, 273, 295.
63

3. Standard Experimental Procedures
Chapter 3
Experimental
The following general experimental techniques were used in this study.
3.1 Chromatographic Methods
3.1.1. Thin-Layer Chromatography
Qualitative thin-layer chromatography was performed on Merck aluminium sheets
(silica gel 60 F254, 0.25 mm). Preparative thin-layer chromatography was
performed on glass plates (20x20 cm), covered with a thin layer (1.0 mm)
Kieselgel PF254 (100 g Kieselgel in 230 mℓ distilled water per 5 plates). The
plates were dried at room temperature and used unactivated. The plates were
loaded to a maximum of 25 mg material per plate. After development the plates
were dried at room temperature in a fast stream of air and the different bands were
distinguished under UV light (254 nm), scraped off and eluted with acetone.
3.1.2. Centrifugal Chromatography
Centrifugal Chromatography was performed on a thin layer of silica gel coated
with a circular glass plate called a rotor. A motor drove the rotor at constant speed
by a shaft passing through a hole in the centre. The compound to be separated was
applied as a solution at the centre of the pre-cast rotor by way of a solvent pump or
hand held syringe. The chosen solvent mixture was then pumped onto the centre.
The solvent is forced by centrifugal forces through the adsorbent layer effectively
separating the individual components as a result of their different affinities for the
silica layer and solvent mixture. A solvent channel collected the eluent and
brought it to the output tube where the fractions were collected.
64

Centrifugal chromatography was performed with an Analtech Cyclograph TM with
commercially available Analtech rotors (4 mm).
3.1.3 Column Chromatography
Separations on Sephadex LH-20 from Pharmacia and Kieselgel from Merck (Art
773, 170-230 mesh) were performed with various column sizes and at differing
flow rates. Fractions were collected in test tubes.
3.1.4 Spraying Reagents
All TLC plates were sprayed with a 2% (v/v) solution of formaldehyde (40%) in
concentrated sulfuric acid and subsequently heated to 110 ºC for maximum colour
development.
3.2 Gas Chromatography
A VARIAN 3300 gas chromatograph was used to record and follow the rate of
reactions.
Column Dimensions:
Column material Silan glass Packing material Chromosorb
Mass 80/100 Liquid phase OV-101
Column length 2 m Internal diameter 3 mm
Rate 30 cm 3 /min Detector: Flame ionization
Injection temperature 200 ºC Column temperature 60-200 ºC
Detector temperature 250 ºC
Conditions:
65

1) Initial column temperature 60 ºC, Initial retention time 0.5 min,
Program 1: Final temperature: 200 ºC, Column 10.0 ºC/min
Column retention time: 0.5 min.
2) Initial column temperature 35 ºC, Initial retention time 0.5 min,
Program 1: Final temperature: 200 ºC, Column 10.0 ºC/min
Column retention time: 0.5 min.
3.3 Spectroscopic Methods
3.3.1 Nuclear Magnetic Resonance Spectroscopy (NMR)
A 600 MHz Bruker spectrometer was used to record the 1 H NMR, COSY, HMQC,
HMBC, 13 C and APT (150 MHz) experiments in either chloroform-d (CDCl3) or
acetone-d6 with TMS (tetramethylsilane) as internal standard. Chemical shifts
were expressed as parts per million (ppm) (δ scale) and coupling constants were
measured in Hz. The following abbreviations of s, d, dd, t, q, m and br are used
to denote singlet, doublet, doublet of doublets, triplet, quartet, multiplet and
broad, respectively.
3.3.2 Mass Spectrometry (MS)
The samples were injected via a Rheodyne valve into a carrier solvent pump at a
flow rate of 50 µl/minute by a Perkin Elmer series 200 micro pump. The sample
was thus delivered into an electrospray ionization source of a Waters Quattro
Ultima triple quadrupole mass spectrometer, operated in the positive or negative
mode. The capillary voltage was 3.5 kV, the source temperature 80 ºC and the
desolvation temperature was 150 ºC. The cone voltage was at 40 V and all other
settings were adjusted for maximum detection of the ions. Data was acquired by
scanning the mass range from mz = 100 to m/z = 1000 at a scan rate of 3
seconds/scan. A representative mass spectrum of the sample was produced by
addition of the spectra across the injection peak and subtraction of the background.
66

3.3.3 Infrared Spectroscopy (IR)
Solid state FR-IR spectra were recorded as KBr pellets on a Bruker Tensor 27
spectrometer in the range of 3000-600 cm -1 .
3.4 Physical Properties Measurement
3.4.1. Melting Point
Melting points were recorded and are uncorrected on a REICHERT, AUSTRIA,
No: 351375.
3.5 Photochemical Reactions
All photochemical reactions were carried out inside the photochemical reactor
RAYON manufactured by SOUTHERN N. E. ULTRAVIOLET Co. Middletown,
Connecticut, USA, equipped with RAYONET PHOTOCHEMICAL REACTOR
lamps CAT. NO. RPR - 3000Å, 3500Å, 2537Å respectively.
3.6 Chemical Methods
3.6.1. Methylation with Diazomethane
Dried material (100 mg) was dissolved in methanol (20 mℓ) and cooled to -10 ºC.
Diazomethane generated by the reaction of NaOH (16 mℓ, 30% in a 95 5 v/v
ethanol solution), with N-methyl-N-nitroso-p-toluenesulphonamide (Diazald, 5 g)
in ether (100 mℓ) and glycol (12.5 mℓ) as co-solvent was distilled slowly into the
sample. The reaction mixture was stored at -10 ºC for 48 hours and the excess
diazomethane was removed at room temperature in a fast moving air current.
67

3.7 Synthesis
3.7.1. a) Synthesis of 2-oxo-N-phenylbutanamide (84)
Thionyl chloride (0.5 mℓ, 6.4 mmol) was added slowly at 0 ºC under a N2
atmosphere to a solution of 2-ketobutyric acid (81) (500 mg, 4.9 mmol) in
anhydrous dichloromethane (10 mℓ). The reaction mixture was left to stir at 0 ºC
for 2 hours after which aniline (82) (0.4 mℓ, 4.4 mmol) was added followed by
TEA (1.4 mℓ, 10.2 mmol) at 0 ºC. The reaction mixture was allowed to attain
room temperature and after 10 minutes the reaction mixture was extracted with
ethyl acetate (200 mℓ) and washed with water. The solvent was then dried over
anhydrous sodium sulfate and concentrated by rotary evaporation. Column
chromatography (silica gel, H:EtOAc:DCM; 9:0.5:0.5 and 1 drop TEA) gave one
fraction, RF 0.10
The fraction RF 0.10 was identified as 2-oxo-N-phenylbutanamide 84 as yellow
crystals, mp 120 ºC, (80 mg, 27%). 1 H NMR: δ (600 MHz, Acetone-d6, Me4Si)
8.76 (1H, NH), 7.65 (2H, d, J = 8.0 Hz, H-2'/ H-6'), 7.37 (2H, t, J = 8.0 Hz, H-3'/
H-5'), 7.18 (1H, t, J = 8.0 Hz, H-4'), 3.05 (2H, q, J = 7.3 Hz, CH2), 1.16 (3H, t, J =
7.3 Hz, CH3). ( 1 H NMR Plate 1a).
13 C NMR: δ (150 MHz, Acetone-d6, Me4Si ) δ 199.9 (C=O), 157.6 (C=O), 136.3
(C-1'), 129.2 (C-3'/ C-5'), 125.2 (C-4'),119.8 (C-2'/ C-6'), 30.0 (-CH2), 7.2 (-CH3) ( 13 C NMR Plate 1b).
IR (KBr): νmax = 3318.4, 2360.2, 1742.2, 1670.4, 1536.1, 1444.3, 1399.9, 1105.8,
753.0 cm -1 ; m/z (ES) 178.08 (100 %, [M+H] + ); Found (ES) [M+H] + , 178.0870
(C10H11NO2 + H + ) requires m/z 178.0868.
68

3.7.1. b) Attempted synthesis of 2-[(4-fluorophenylsulfonyl)methyl]-2-
hydroxy-N-phenylbutanamide (85)
4-Fluorophenyl methyl sulphone 14 (164.0 mg, 0.9 mmol) was dissolved in
anhydrous THF (10 mℓ) and the temperature was dropped to -75 ºC at which time
BuLi (0.1 ml, 0.9 mmol) was added, in a N2 atmosphere.The reaction mixture was
allowed to reach room temperature and stirred for 1 hour at room temperature.
After cooling to -75 ºC again, 2-oxo-N-phenylbutanamide 84 (83.9 mg, 0.47
mmol) was dissolved in anhydrous THF (2 mℓ) and added to the reaction mixture.
The mixture was left overnight to attain room temperature and stirred at this
temperature. NH4Cl was added to the mixture and dried under N2. Column
chromatography (silica gel, H:EtOAc:DCM; 4:3:3 and 1 drop TEA) gave two
fractions, RF 0.60 and RF 0.40.
The fraction RF 0.60 yielded unreacted 2-oxo-N-phenylbutanamide 84 as yellow
crystals, mp 120 ºC, (80 mg). NMR identical to plate 1.
The fraction RF 0.40 yielded unreacted 4-fluorophenyl methyl sulfone 14 as a
white amorphous solid (150 mg).
1 H NMR: δ (600 MHz, Acetone-d6, Me4Si ) 7.9 (dd, J = ,5.2 Hz, 8.9 Hz, H-2/ H-
6), 7.3 (t, J = 8.9 Hz, H-3/ H-5), 3.0 (1H, s,CH 3). ( 1 H NMR plate 10a).
13 C NMR: δ (150 MHz, Acetone-d6, Me4Si ) 166.4 (d, J = ,C-4), 137.8 (d, J = 3Hz
, C-1), 130.4 (d, J = 9.7Hz, C-2/ 6), 116.3 (d, J = 22.8Hz, C-3/ 5), 43.6 (-CH3). ( 13 C NMR plate 10b).
19 F NMR: δ (564.2MHz, Acetone-d6, Trifluoroacetic acid) -106.7 (F). ( 19 F NMR
plate 10c).
69

3.7.2. a) N-[4-Cyano-3-(trifluoromethyl)phenyl]-2-oxobutanamide (86)
Thionyl chloride (1 mℓ, 13.7 mmol) was added slowly at 0 ºC under a N2
atmosphere to a solution of ketobutyric acid 81 (100 mg, 0.98 mmol) in anhydrous
dichloromethane (10 mℓ). The reaction mixture was left to stir at 0 ºC for 2 hours
after which 4-amino-3-(trifluoromethyl)benzonitrile 6 (150 mg, 0.8 mmol) was
added followed by TEA (2.7 mℓ, 19.6 mmol) at 0 ºC. The reaction mixture was
allowed to attain room temperature and after 10 minutes the reaction mixture was
extracted with ethyl acetate (200 mℓ) and washed with water. The solvent was
dried over anhydrous sodium sulfate and concentrated by rotary evaporation.
Column chromatography (silica gel, H:EtOAc:DCM; 4:3:3 and 1 drop TEA) gave
one fraction, RF 0.60.
The fraction RF 0.60 was identified as N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
oxobutanamide 86, orange crystals, mp 120 ºC, (80 mg, 36 %). 1 H NMR: δ (600
MHz, Acetone-d6, Me4Si) 10.30 (1H, s, NH), 8.55 (1H, d, J = 2.0 Hz, H-2'), 8.37
(1H, dd, J = 1.9 Hz, 8.6 Hz, H-6'), 8.09 (1H, d, J = 8.6 Hz, H-5'), 3.02 (2H, q, J =
7.18 Hz, CH2), 1.10 (3H, t, J = 7.16 Hz, CH3). ( 1 H NMR plate 2a).
13 C NMR (150 MHz, Acetone-d6, Me4Si ): δ 159.5 (C=O), 142.4 (C-1'), 136.3 (C-
5'), 132.8 (q, 2 JFC = 34.6 Hz, C-3'), 123.0 (C-6'),122.6 (q, 1 JFC = 273.5 Hz, CF3), 117.8 (q, 3 J FC = 5.4 Hz, C-2'), 115.3 (C≡N), 104.1 (d, 3 J FC = 2.26 Hz, C-4), 29.6
(CH2), 6.4 (CH3). ( 13 C NMR Plate 2b).
19 F NMR δ: (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -62.63 (CF 3). ( 19 F
NMR plate 2c).
IR (KBr): νmax = 3333.6, 2232.4, 1703.1, 1585.8, 1434.7, 1324.2, 1181.3, 1097.4
cm -1 ; m/z (ES) 269 (100 %), [M-H + ] - ); Found (ES) [M-H] - , 269.0541
(C12H9F3N2O2 - H + ) requires m/z 269.0538.
70

3.7.2. b) Attempted synthesis of N-(4-cyano-3-(trifluoromethyl)phenyl)-2-[(4-
fluorophenylsulfonyl)methyl]-2-hydroxybutanamide (85)
4-Fluorophenyl methyl sulphone 14 (164.0 mg, 0.9 mmol) was dissolved in
anhydrous THF (10 mℓ) and cooled to -75 ºC at which time BuLi (0.1 mℓ, 0.9
mmol) was added (under N2 atmosphere). The reaction mixture was allowed to
reach room temperature and stirred for 1 hour at room temperature. N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-oxobutanamide 86, (83.9 mg, 0.5 mmol) was dissolved
in anhydrous THF (2 mℓ) and added to the reaction mixture at -75 ºC. The reaction
mixture was left overnight to attain room temperature and stirred at this
temperature. The reaction mixture was divided and NH4Cl was added to one half
and the other half was dried under N2. Column chromatography (silica gel,
H:EtOAc:DCM; 4:3:3 and 1 drop TEA) gave two fractions, RF 0.60 and RF 0.40.
The fraction RF 0.60 yielded amide 86 as orange crystals, mp 120 ºC, (80 mg).
NMR identical to Plate 2.
The fraction RF 0.40 yielded 4-fluorophenyl methyl sulfone (14) as a white
amorphous solid, (160 mg). NMR identical to Plate 10.
3.7.3. Extraction of bicalutamide; N-[4-cyano-3-(trifluoromethyl)phenyl]-3-
[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide (1) from
Casodex ®
A Casodex ® tablet (50 mg) was crushed, extracted with acetone and concentration
by rotary evaporation gave 48.9 mg of bicalutamide (1).
1 H NMR: δ (600 MHz, Acetone-d6) 9.84 (1H, NH), 8.43 (1H, d, J = 2.0 Hz, H-2"),
8.08 (1H, dd, J = 8.56 Hz, 2.0 Hz, H-6"), 7.89 (1H, d, J = 8.56 Hz, H-5"), 7.86
(2H, dd, J = 9.0 Hz, 5.0 Hz, H-2'/ 6'), 7.16 (2H, t, J = 9.0 Hz, H-3'/ 5'), 4.0 (1H, d,
J = 14.7, H-3), 3.57 (1H, d, J = 14.7, H-3), 1.42 (3H, s, CH3).( 1 H NMR Plate 3a).
71

13 C NMR: δ (150MHz, Acetone-d6) 172.6 (amide C=O), 165.0 (d, 1 J FC = 253.3, C-
4'),142.7 (C–1"), 136.9 (d, 1 JFC = 2.0 Hz, C–1'), 135.5 (C-5"), 132.0 (q, 2 JFC =
29.41, C-3"), 131.1 (d, 4 JFC = 9.67 Hz, C-2'/ 6'), 122.3 (q, 1 JFC = 273.77, -CF3), 122.29 (C-6"), 117.0 (q, 3 JFC = 5.42 Hz, C-2"), 115.5 (d, 2 JFC = 22.9 Hz, C-3'/ 5'),
114.9 (C≡N), 102.9 (d, J = 2.2 Hz, C-4"), 73.3 (C-2), 62.7 (CH 2), 26.5 (CH 3).( 13 C
NMR Plate 3b).
19 F NMR: δ (564.2MHz, Acetone-d6, Trifluoroacetic acid) -62.60 (CF3), -106.50
(F). ( 19 F NMR Plate 3c).
IR (KBr): νmax = 3338.2, 2230.2, 1689.2, 1516.78, 1328.1, 1328.1, 1290.4, 1179.5,
1140.8 cm -1 ; M + 430.38.
3.7.4. 3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methyl-N-[4-methyl-3-
(trifluoromethyl)phenyl]propanamide (94)
Bicalutamide (1) (50 mg, 0.2 mmol) was dissolved in ethanol (20 mℓ) and equal
equivalents palladium on activated charcoal was added. The reaction mixture was
stirred at room temperature under H2 gas for 24 hours. The catalyst was filtered off
and the solvent was evaporated under reduced pressure. Column chromatography
(silica gel, H:EtOAc and 1 drop TEA 5:5) yielded two fractions, RF 0.36 and RF
0.18.
The fraction RF 0.18 yielded unreacted bicalutamide (1) as white crystals from
ethyl acetate and petroleum ether from acetone, mp 191-193 ºC, (20 mg). NMR
identical to plate 3.
The fraction RF 0.36 yielded 3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-N-
[4-methyl-3-(trifluoromethyl)phenyl]propanamide (94) as a white amorphous
solid, (15 mg, 50 %). 1 H NMR: δ (600 MHz, CDCl3, Me4Si ) 8.74 (1H, NH) 7.87
(2H, dd, J = 4.97 Hz, 8.88 Hz, H-2'/ 6'), 7.68 (1H, d, J = 2.30 Hz, H-2"), 7.46 (1H,
dd, J = 2.30 Hz, 8.37 Hz, H-6"), 7.24 (1H, d, J = 8.37 Hz, H-5"), 7.11 (2H, t, J =
8.88 Hz, H-3'/ 5'), 4.02 (1H, d, J = 14.2 Hz, H-3), 3.48 (1H, d, J = 14.2 Hz, H-3),
2.45 (3H, d, J = 1.38 Hz, CH3), 1.59 (3H, s, -CH3). ( 1 H NMR Plate 4a).
72

13 C NMR: δ (150 MHz, CDCl3 ,Me4Si ) 170.4 (amide C=O), 166.2 (d, 2 JFC = 258.3
Hz, C-4'), 135 (d, 1 JFC = 3.1 Hz, C-1'), 134.8 (C-1"), 132.9 (C-5"), 130.9 (d, 4 JFC =
9.8 Hz, C-2'/ 6'), 129.4 (q, 1 JFC = 30.1 Hz, C-3"), 125.0 (q, 1 JFC = 273.02 Hz, CF3),
122.3 (C-6"), 117,0 (q, 3 JFC = 5.8 Hz, C-2"), 116.7 (d, 2 JFC = 22.8 Hz, C-3'/ 5'),
74.2 (C-2), 61.6 (CH2), 27.8 (CH3), 18.8 (CH3). ( 13 C NMR Plate 4b).
19 F NMR: δ (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -61.40 (CF3), -106.60
(F). ( 19 F NMR Plate 4c).
IR (KBr): νmax = 3362.2, 1530.2, 1320.4, 1281.4, 1137.3, 835.0 cm -1 ; m/z (ES) 420
(100 %, [M+H] + ); Found (ES) [M+H] + , 420.0895 (C18H17NO4F4S + H + ) requires
m/z 420.0893.
3.7.5. Synthesis of (3-(4-fluorophenylsulfonyl)-2-hydroxy-2methylpropanamido)-2-(trifluoromethyl)benzamide
(96)
Bicalutamide (1) (50 mg, 0.2 mmol) was refluxed in benzene (5 mℓ) with equal
equivalents of ρ-toluenesulfonic acid and a few drops of H2SO4 for 5 hours.
Column chromatography (H:EtOAc and 1 drop TEA; 5:5) gave two fractions RF 0.60 and RF 0.30.
The fraction R F 0.6 yielded unreacted bicalutamide (1) as white crystals from ethyl
acetate and petroleum ether from acetone, mp 191-193 ºC, (5 mg). NMR identical
to plate 3.
The fraction R F 0.3 yielded 4-[3-(4-fluorophenylsulfonyl)-2-hydroxy-2-
methylpropanamido]-2-(trifluoromethyl)benzamide (96) as a white amorphous
solid, (20 mg, 44 %). 1 H NMR: δ (600 MHz, Acetone-d6, Me4Si ) 9.66 (1H, OH)
8,21 (2H, d, J = 2.1 Hz, H-3"), 8.0 (3H, m, H-2'/ H-6'/ H-5"), 7.58 (1H, d, J = 8.3
Hz, H-6"), 7.30 (2H, t, J = 8.7 Hz, H-3'/ 5'), 4.06 (1H, d, J = 14.8 Hz, H-3), 3.68
(1H, d, J = 14.8 Hz, H-3), 1.55 (3H, s, CH3).( 1 H NMR Plate 5a).
13 C NMR: δ (150 MHz, Acetone-d6, Me4Si ) 171.7 (C=O), 168.0 (C=O), 164.9 (d,
2
JFC = 253.1 Hz, C-4'), 138.9 (C-4"), 137.6 (d, 1 JFC = 3.0 Hz, C-1'), 132.9 (C-6"),
73

130.8 (d, 4 JFC = 9.60 Hz, C-2'/ 6'), 127.5 (C-1"), 126.6 (q, 1 JFC = 34.6 Hz, C-2"),
124.1 (q, 1 JFC = 273.7 Hz, CF3), 122.5 (C-5"), 116,7 (q, 3 JFC = 5.5 Hz, C-3"), 115.2
(d, 2 JFC = 22.9 Hz, C-3'/ 5'), 73.0 (C-2), 62.5 (CH2), 26.3 (CH3). ( 13 C NMR Plate
5b)
19 F NMR: δ (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -59.63 (CF3), -106.80
(F) ( 19 F NMR Plate 5c).
IR (KBr): νmax = 3447.7, 2964.0, 2925.8, 1673.38, 1494.9, 1142.36 cm -1 ; m/z (ES)
447 (100 %, [M-H + ] - ; Found (ES) [M-H + ] - , 447.0640 (C18H17NO4F4S - H + )
requires m/z 447.0638.
3.7.6. Attempted synthesis of (Z)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4fluorophenylsulfonyl)-2-methylacrylamide
(99)
Bicalutamide (50 mg, 0.1 mmol) was dissolved in THF (5 mℓ). LDA (0.1 mℓ) was
added and the reaction mixture was then heated at 100 ºC in an autoclave under N2 (1 bar) for 2 hours. The reaction mixture was allowed to cool overnight. Column
chromatography (T:A; 8:2) gave three fractions RF 0.70, RF 0.60 and RF 0.30.
The fraction R F 0.30 yielded unreacted bicalutamide (1) as white crystals from
ethyl acetate and petroleum ether from acetone, mp 191-193 ºC (20 mg). NMR
identical to plate 3.
The fraction RF 0.60 yielded 4-fluorophenyl methyl sulfone (14) as a white
amorphous solid, (10 mg).
1 H NMR: δ (600 MHz, CDCl3, Me4Si ) 7.98 (dd, 2H, J = 5.2 Hz, 8.9 Hz, H-3/ H-
5), 7.26 (t, 2H, J = 8.9 Hz, H-3/ H-5), 3.07 (CH3). NMR identical to plate 10.
The fraction RF 0.70 yielded 4-amino-3-(trifluoromethyl)benzonitrile (6) as an
orange amorphous solid, (5 mg). NMR identical to plate 9.
74

3.7.7. Synthesis of 1-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(4-
fluorophenylsulfonyl)-2-methyl-1-oxopropan-2-yl 3-nitrobenzoate (98) and
Synthesis of (Z)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-
fluorophenylsulfonyl)-2-methylacrylamide (99)
Bicalutamide (1) (100 mg, 0.23 mmol) was dissolved in dichloromethane (10 mℓ)
and pyridine (0.5 mℓ) and a solution of 3-nitrobenzoyl chloride (97) (100 mg, 0.4
mmol) in dichloromethane (0.5 mℓ) was added, the mixture was cooled to 0 ºC,
followed by the addition of 4-dimethylaminopyridine (50 mg, 0.4 mmol). The
reaction mixture was allowed to attain room temperature after which it was heated
for 2 hours at 40 ºC and subsequently stirred for five days at room temperature.
The reaction mixture was dried under N2 and column chromatography (silica gel,
H:EtOAc; 5:5 and 1 drop aq. NH3) gave three fractions, RF 0.60, RF 0.40 and RF
0.27.
The fraction RF 0.27 yielded unreacted bicalutamide (1) as white crystals from
ethyl acetate and petroleum ether from acetone, mp 191-193 ºC, (10 mg,). NMR
identical to plate 3.
The fraction RF 0.40 yielded 1-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(4fluorophenylsulfonyl)-2-methyl-1-oxopropan-2-yl
3-nitrobenzoate (98) as a
yellow-white amorphous solid, (20 mg, 22 %).
1
H NMR: δ (600 MHz, acetone-d6) 9.93 (s, NH), 8.58 (ddd, J = 0.4, 1.6, 2.3 Hz,
H-2'''), 8.42 (ddd, 1H, J = 1.1, 2.3, 8.2 Hz, H-4'''), 8.25 (ddd, 1H, J = 1.1 Hz, 1.6
Hz, 7.5 Hz, H-6'''), 8.11 (d, J = 2.0 Hz, H-2"), 7.96 (dd, J = 2.0 Hz, 8.8 Hz, H-6" ),
7.9-7.8 (m, H-5"/ H-3'/ H-5'), 7.74 (ddd, J = 0.4 Hz, 7.5 Hz, 8.2 Hz, H-5'''), 7.29 (t,
J = 8.8 Hz, H-2'/ H-6') , 4.36 (d, J = 15.3 Hz, H-3), 4.12 (d, J = 15.3 Hz, H-3 ), 2.0
(s, CH3). ( 1 H NMR Plate 6a).
13 C NMR: δ (150 MHz, acetone-d6) 168.9 (C=O), 165.3 (d, 1 J FC = 254.2 Hz, C-4'),
162.9 (C=O), 147.8 (C-1'''), 142.26 (C–1"), 136.5 (d, 1 J FC = 3.4 Hz, C-1'), 135.3
(C-5''), 135.6 (C-6'"), 135.3 (q, 2 J FC = 32.0 Hz, C-3"), 130.8 (d, 4 J CF = 9.8 Hz , C-
2'/ C-6'), 129.8 (C-5'''), 127.5 (C-4'''), 24.0 (C-2'''), 123.2 (C-6"), 122.6 (q, 2 J FC =
75

272.2 Hz, C-3"), 122.1 (q, 1 JFC = 273.8, CF3) 117.9 (q, 1 JFC = 4.6 Hz, C-2"), 116.5
(d, 1 JFC = 22.8 Hz, C-3'/ 5'), 115.2 (C≡N), 103.5 (C-4"), 80.24 (C-2), 57.0 (CH2), 22.4 (CH3). ( 13 C NMR Plate 6b).
19 F NMR: δ (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -62.70(CF3), -106.0
(F).( 13 C NMR Plate 6c)
IR (KBr): νmax = 3430.0, 3105.7, 2933.3, 2360.3, 2231,9, 1735.9, 1592.1, 1535.1,
1327.02, 1144.29, 842.2 cm -1 ; m/z (ES) 579 (100 %, [M-H] - ); Found (ES) [M-H] - ,
579.0650 (C25H17F4N3O2S - H + ) requires m/z 579.0645.
The fraction RF 0.60 yielded (Z)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-
Fluorophenylsulfonyl)-2-methylacylamide (99) as a white amorphous solid, (31.8
mg, 35.3 %). 1 H NMR: δ (600 MHz, Acetone-d6) 10.14 (1H, OH), 8.30 (d, 1H, J =
1.9 Hz, H-2"), 8.14 (dd, 1H, J = 1.9 Hz, 8.6 Hz, H-6"), 8.08 (dd, 2H, J = 5.3 Hz,
8.9 Hz, H-2'/ 6'), 8.02 (d, J = 8.6 Hz, H-5"), 7.47 (t, 2H, J = 8.9 Hz, H-3'/ 5'), 7.22
(q, 1H, J = 1.5 Hz, C-3), 2.41 (d, 3H, J = 1.5 Hz, CH3). ( 1 H NMR Plate 7a).
13 C NMR: δ (150 MHz, Acetone-d6) 167.7 (C=O), 166.5 (d, 1 J FC = 163.2 Hz, C-
4'), 147.1 (C-2), 143.9 (C-1"), 138.5 (d, 1 JFC = 3.2 Hz, C-1'), 137.0 (C-5"), 134.2
(C-3), 133.5 (q, 2 JFC = 32.0 Hz, C-3"), 131.7 (d, 4 JFC = 10.0 Hz, C-2'/ 6'), 123.9 (C-
6"), 123.5 (q, 1 JFC = 273.2 Hz, CF3), 118.7 (q, 3 JFC = 5.2 Hz, C-2"),117.7 (d, 2 JFC =
2.3 Hz, H-3'/ 5'), 116.2 (CN), 104.8 (d, 1 JFC = 2.4 Hz, C-4"), 14.2 (CH3). ( 13 C
NMR Plate 7b).
19 F NMR: δ (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -62.30 (CF3), -105.50
(F). ( 19 F NMR Plate 7c).
IR (KBr): νmax = 3320.2, 2231.5, 1697.03, 1592.4, 1533.2, 1328.4, 1146.0, 840.0
cm -1 ; m/z (ES) 413 (100 %, [M+H] + ); Found (ES) [M+H] + , 413.0587
(C18H12F4N2O3S+ H + ) requires m/z 413.0583.
76

3.7.8 Synthesis of 4-amino-2-ethoxybenzonitrile (100) (I)
Bicalutamide (1) (100 mg, 0.16 mmol) was dissolved in ethanol (100 mℓ) and
irradiated for 24 hours at 300 nm under a N2 atmosphere. The solvent was
concentrated by rotary evaporation. Column chromatography (silica gel,
H:EtOAc:DCM and 1 drop TEA, 4:3:3 ) yielded three fractions RF 0.50, RF 0.38
and RF 0.17.
The fraction RF 0.17 yielded unreacted bicalutamide (1) as white crystals from
ethyl acetate and petroleum ether from acetone, mp 191-193 ºC, (34.7 mg). NMR
identical to plate 3.
The fraction RF 0.38 yielded 4-amino-2-ethoxybenzonitrile (100) as a yellow
amorphous solid, (20 mg, 30.6 %). 1 H NMR: δ (600 MHz, acetone-d6) 7.52 (1H,
d, J = 8.5 Hz, H-5), 7.36 (1H, d, J = 2.5 Hz, H-2), 6.94 (1H, dd, J = 2.5 Hz, , H-6),
5.86, (2H, NH2) 4.37 (2H, dd, J = 7.1 Hz, 14.3 Hz, CH2), 1.38 (3H, t, J = 7.1Hz,
CH3). ( 1 H NMR Plate 8a).
13 C NMR: δ (150 MHz, acetone-d6) 164.3 (C-2), 152.3 (C-3), 136.0 (C–5), 134.0
(C–4), 118.4 (CN), 116.6 (C-6), 115.5 (C-6), 98.1 (C-NH2), 61.3 (CH2), 13.5
(CH3). ( 13 C NMR plate 8b).
IR (KBr): υ 3458.1, 3372.6, 3234.2, 2207.7, 1705.4, 1601.7, 1267.8, 1247.5 cm -1 ;
m/z (ES) 163 (100 %, [M+H] + ); Found (ES) [M+H] + , 163.0874 (C9H10N2O + H + )
requires m/z 163.0872.
The fraction RF 0.5 yielded 4-amino-3-(trifluoromethyl)benzonitrile (6) as an
orange amorphous solid, (20 mg).
1 H NMR: δ (600 MHz, Acetone-d6, Me4Si) 7.6 (1H, d, J = 8.6 Hz, H-5), 8.4 (1H,
d, J = 2.3Hz, 8.6Hz, H-2), 7.0 (1H, dd, J = 2.3Hz, 8.5Hz, H-5), 6.1 (2H, s, NH2). ( 1 H NMR plate 9a).
13 C NMR: δ (150 MHz, Acetone-d6, Me4Si ) 152.8 (C-4), 136.3 (C-6), 133.2 (q,
2 JFC = 31.7 Hz, C-3), 122.2 (q, 1 J FC = 272.9 Hz, CF 3 ), 116.7 (q, 3 J FC = 4.9 Hz, C-
77

3), 111.3 (d, 3 J FC = 2.1 Hz, C-5), 111.24 (CN), 94.1 (C-4 and C-NH 2). ( 13 C NMR
plate 9b).
19 F NMR: δ (564.2 MHz, Acetone-d6, Trifluoroacetic acid) -63.0 (CF 3). ( 19 F NMR
plate 9c).
3.7.9. Synthesis of 4-amino-2-ethoxybenzonitrile (100) (II)
4-Amino-2-(trifluoromethyl)benzonitrile (6) (200 mg, 1.1 mmol) was dissolved in
ethanol (100 mℓ) and irradiated for 24 hours at 300 nm under a N2 atmosphere.
The solvent was concentrated by rotary evaporation. Column chromatography
(silica gel, H:EtOAc and 1 drop TEA, 5:5 ) yielded two fractions RF 0.50 and RF
0.38.
The fraction RF 0.38 yielded 4-amino-2-ethoxybenzonitrile (100) as a yellow
amorphous solid, (100 mg, 83 %). NMR identical to Plate 8.
The fraction RF 0.5 yielded unreacted 4-amino-2-(trifluoromethyl)benzonitrile (6)
as a orange amorphous solid, (80 mg). NMR identical to Plate 9.
78